Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

ABSTRACT

The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using them.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/644,072, filed Oct. 3, 2012, entitled Modified Nucleosides,Nucleotides, and Nucleic Acids, and Uses Thereof which claims priorityto U.S. Provisional Patent Application No. 61/542,533, filed Oct. 3,2011, entitled Modified Nucleosides, Nucleotides, and Nucleic Acids, andUses Thereof, the contents of each are herein incorporated by referencein their entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file, entitled M009SQLST.txt,was created on Jan. 17, 2013 and is 9,970 bytes in size. The informationin electronic format of the Sequence Listing is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present disclosure provides compositions and methods using modifiednucleic acids to modulate cellular function. The modified nucleic acidsof the invention may encode peptides, polypeptides or multiple proteins.The encoded molecules may be used as therapeutics and/or diagnostics.

BACKGROUND OF THE INVENTION

Naturally occurring RNAs are synthesized from four basicribonucleotides: ATP, CTP, UTP and GTP, but may containpost-transcriptionally modified nucleotides. Further, approximately onehundred different nucleoside modifications have been identified in RNA(Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA ModificationDatabase: 1999 update. Nucl Acids Res 27: 196-197). The role ofnucleoside modifications on the immune-stimulatory potential and on thetranslation efficiency of RNA, however, is unclear.

There are multiple problems with prior methodologies of effectingprotein expression. For example, heterologous DNA introduced into a cellcan be inherited by daughter cells (whether or not the heterologous DNAhas integrated into the chromosome) or by offspring. Introduced DNA canintegrate into host cell genomic DNA at some frequency, resulting inalterations and/or damage to the host cell genomic DNA. In addition,multiple steps must occur before a protein is made. Once inside thecell, DNA must be transported into the nucleus where it is transcribedinto RNA. The RNA transcribed from DNA must then enter the cytoplasmwhere it is translated into protein. This need for multiple processingsteps creates lag times before the generation of a protein of interest.Further, it is difficult to obtain DNA expression in cells; frequentlyDNA enters cells but is not expressed or not expressed at reasonablerates or concentrations. This can be a particular problem when DNA isintroduced into cells such as primary cells or modified cell lines.

There is a need in the art for biological modalities to address themodulation of intracellular translation of nucleic acids.

SUMMARY OF THE INVENTION

The present disclosure provides, inter alia, modified nucleosides,modified nucleotides, and modified nucleic acids which can exhibit areduced innate immune response when introduced into a population ofcells, both in vivo and ex vivo.

The present invention provides polynucleotides which may be isolated orpurified. These polynucleotides may encode one or more polypeptides ofinterest and comprise a sequence of n number of linked nucleosides ornucleotides comprising at least one modified nucleoside or nucleotide ascompared to the chemical structure of an A, G, U or C nucleoside ornucleotide. The polynucleotides may also contain a 5′ UTR comprising atleast one Kozak sequence, a 3′ UTR, and at least one 5′ cap structure.The isolated polynucleotides may further contain a poly-A tail and maybe purified.

The isolated polynucleotides of the invention also comprise at least one5′ cap structure selected from the group consisting of Cap0, Cap 1,ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

Modifications of the polynucleotides of the invention may be on thenucleoside base and/or sugar portion of the nucleosides which comprisethe polynucleotide.

In some embodiments, the modification is on the nucleobase and isselected from the group consisting of pseudouridine orN1-methylpseudouridine.

In some embodiments, the modified nucleoside is not pseudouridine (ψ) or5-methyl-cytidine (m5C).

In some embodiments, multiple modifications are included in the modifiednucleic acid or in one or more individual nucleoside or nucleotide. Forexample, modifications to a nucleoside may include one or moremodifications to the nucleobase and the sugar.

In some embodiments are provided novel building blocks, e.g.,nucleosides and nucleotides for the preparation of modifiedpolynucleotides and their method of synthesis and manufacture.

The present invention also provides for pharmaceutical compositionscomprising the modified polynucleotides described herein. These may alsofurther include one or more pharmaceutically acceptable excipientsselected from a solvent, aqueous solvent, non-aqueous solvent,dispersion media, diluent, dispersion, suspension aid, surface activeagent, isotonic agent, thickening or emulsifying agent, preservative,lipid, lipidoids liposome, lipid nanoparticle, core-shell nanoparticles,polymer, lipoplexe peptide, protein, cell, hyaluronidase, and mixturesthereof.

Methods of using the polynucleotides and modified nucleic acids of theinvention are also provided. In this instance, the polynucleotides maybe formulated by any means known in the art or administered via any ofseveral routes including injection by intradermal, subcutaneous orintramuscular means.

Administration of the modified nucleic acids of the invention may be viatwo or more equal or unequal split doses. In some embodiments, the levelof the polypeptide produced by the subject by administering split dosesof the polynucleotide is greater than the levels produced byadministering the same total daily dose of polynucleotide as a singleadministration.

Detection of the modified nucleic acids or the encoded polypeptides maybe performed in the bodily fluid of the subject or patient where thebodily fluid is selected from the group consisting of peripheral blood,serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood.

In some embodiments, administration is according to a dosing regimenwhich occurs over the course of hours, days, weeks, months, or years andmay be achieved by using one or more devices selected from multi-needleinjection systems, catheter or lumen systems, and ultrasound, electricalor radiation based systems.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the present disclosure will be apparentfrom the following detailed description and figures, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 provides the spectrum and graphs of the analytical results forN4-Me-CTP (NTP of compound 1). FIG. 1A provides the nuclear magneticresonance (NMR) spectrum in DMSO and FIG. 1B provides the NMR spectrumin D₂O, FIG. 1C provides the mass spectrometry (MS) results, and FIG. 1Dis the high performance liquid chromatography (HPLC) results forN4-methylcytidine (N4-Me-cytidine, compound 1).

FIG. 2 shows the HPLC results for N4-Me-CTP (NTP of compound 1).

FIG. 3 provides the analytical results for 2′-OMe-N,N-di-Me-CTP (NTP ofcompound 2). FIG. 3A provides the NMR spectrum. FIG. 3B provides the MSresults. FIG. 3C provides HPLC results for2′-O-methyl-N⁴,N⁴-dimethylcytidine (2′-OMe-N,N-di-Me-cytidine, compound2).

FIG. 4 shows the HPLC results for 2′-OMe-N,N-di-Me-CTP (NTP of compound2).

FIG. 5 provides the HPLC results for 5-methoxycarbonylmethoxy-UTP (NTPof compound 3).

FIG. 6 provides the analytical results of 3-methyl pseudouridine(compound 4). FIG. 6A provides the NMR spectrum of 3-methylpseudouridine (compound 4) and FIG. 6B provides the HPLC results for3-methyl pseudouridine (compound 4).

FIG. 7 provides the analytical results of 5-TBDMS-OCH₂-cytidine(compound 6). FIG. 7A provide the NMR spectrum, FIG. 7B provides the MSresults, and FIG. 7C provides the HPLC results for 5-TBDMS-OCH₂-cytidine(compound 6).

FIG. 8 provides the analytical results of 5-trifluoromethyl uridine(compound 8). FIG. 8A provides the NMR spectrum, FIG. 8B provides MSresults, and FIG. 8C provides HPLC results for 5-trifluoromethyl uridine(compound 8).

FIG. 9 provides the NMR spectrum results for of5-(methoxycarbonyl)methyl uridine (compound 9).

FIG. 10 provides a graph showing the variability of protein (GCSF; lineB) and cytokine (interferon-alpha (IFNa); line A and tumor necrosisfactor-alpha (TNFa); line C) expression as function of percentmodification.

DETAILED DESCRIPTION

The present disclosure provides, inter alia, modified nucleosides,modified nucleotides, and modified nucleic acids that exhibit improvedtherapeutic properties including, but not limited to, a reduced innateimmune response when introduced into a population of cells.

As there remains a need in the art for therapeutic modalities to addressthe myriad of barriers surrounding the efficacious modulation ofintracellular translation and processing of nucleic acids encodingpolypeptides or fragments thereof, the inventors have shown that certainmodified mRNA sequences have the potential as therapeutics with benefitsbeyond just evading, avoiding or diminishing the immune response.

The present invention addresses this need by providing nucleic acidbased compounds or polynucleotides which encode a polypeptide ofinterest (e.g., modified mRNA) and which have structural and/or chemicalfeatures that avoid one or more of the problems in the art, for example,features which are useful for optimizing nucleic acid-based therapeuticswhile retaining structural and functional integrity, overcoming thethreshold of expression, improving expression rates, half life and/orprotein concentrations, optimizing protein localization, and avoidingdeleterious bio-responses such as the immune response and/or degradationpathways.

Provided herein, in part, are polynucleotides encoding polypeptides ofinterest which have been chemically modified to improve one or more ofthe stability and/or clearance in tissues, receptor uptake and/orkinetics, cellular access by the compositions, engagement withtranslational machinery, mRNA half-life, translation efficiency, immuneevasion, protein production capacity, secretion efficiency (whenapplicable), accessibility to circulation, protein half-life and/ormodulation of a cell's status, function and/or activity.

The modified nucleosides, nucleotides and nucleic acids of theinvention, including the combination of modifications taught herein havesuperior properties making them more suitable as therapeutic modalities.

It has been determined that the “all or none” model in the art is sorelyinsufficient to describe the biological phenomena associated with thetherapeutic utility of modified mRNA. The present inventors havedetermined that to improve protein production, one may consider thenature of the modification, or combination of modifications, the percentmodification and survey more than one cytokine or metric to determinethe efficacy and risk profile of a particular modified mRNA.

In one aspect of the invention, methods of determining the effectivenessof a modified mRNA as compared to unmodified involves the measure andanalysis of one or more cytokines whose expression is triggered by theadministration of the exogenous nucleic acid of the invention. Thesevalues are compared to administration of an unmodified nucleic acid orto a standard metric such as cytokine response, PolyIC, R-848 or otherstandard known in the art.

One example of a standard metric developed herein is the measure of theratio of the level or amount of encoded polypeptide (protein) producedin the cell, tissue or organism to the level or amount of one or more(or a panel) of cytokines whose expression is triggered in the cell,tissue or organism as a result of administration or contact with themodified nucleic acid. Such ratios are referred to herein as theProtein:Cytokine Ratio or “PC” Ratio. The higher the PC ratio, the moreefficacious the modified nucleic acid (polynucleotide encoding theprotein measured). Preferred PC Ratios, by cytokine, of the presentinvention may be greater than 1, greater than 10, greater than 100,greater than 1000, greater than 10,000 or more. Modified nucleic acidshaving higher PC Ratios than a modified nucleic acid of a different orunmodified construct are preferred.

The PC ratio may be further qualified by the percent modificationpresent in the polynucleotide. For example, normalized to a 100%modified nucleic acid, the protein production as a function of cytokine(or risk) or cytokine profile can be determined.

In one embodiment, the present invention provides a method fordetermining, across chemistries, cytokines or percent modification, therelative efficacy of any particular modified polynucleotide by comparingthe PC Ratio of the modified nucleic acid (polynucleotide).

In another embodiment, the chemically modified mRNA are substantiallynon toxic and non mutagenic.

In one embodiment, the modified nucleosides, modified nucleotides, andmodified nucleic acids can be chemically modified on the major grooveface, thereby disrupting major groove binding partner interactions,which may cause innate immune responses. Further, these modifiednucleosides, modified nucleotides, and modified nucleic acids can beused to deliver a payload, e.g., detectable or therapeutic agent, to abiological target. For example, the nucleic acids can be covalentlylinked to a payload, e.g. a detectable or therapeutic agent, through alinker attached to the nucleobase or the sugar moiety. The compositionsand methods described herein can be used, in vivo and in vitro, bothextracellarly or intracellularly, as well as in assays such as cell freeassays.

In some embodiments, the present disclosure provides compoundscomprising a nucleotide that disrupts binding of a major grooveinteracting, e.g. binding, partner with a nucleic acid, wherein thenucleotide has decreased binding affinity to major groove interactingpartners.

In another aspect, the present disclosure provides nucleotides thatcontain chemical modifications, wherein the nucleotide has alteredbinding to major groove interacting partners.

In some embodiments, the chemical modifications are located on the majorgroove face of the nucleobase, and wherein the chemical modificationscan include replacing or substituting an atom of a pyrimidine nucleobasewith an amine, an SH, an alkyl (e.g., methyl or ethyl), or a halo (e.g.,chloro or fluoro).

In another aspect, the present disclosure provides chemicalmodifications located on the sugar moiety of the nucleotide.

In another aspect, the present disclosure provides chemicalmodifications located on the phosphate backbone of the nucleic acid.

In some embodiments, the chemical modifications alter theelectrochemistry on the major groove face of the nucleic acid.

In another aspect, the present disclosure provides nucleotides thatcontain chemical modifications, wherein the nucleotide reduces thecellular innate immune response, as compared to the cellular innateimmune induced by a corresponding unmodified nucleic acid.

In another aspect, the present disclosure provides nucleic acidsequences comprising at least two nucleotides, the nucleic acid sequencecomprising a nucleotide that disrupts binding of a major grooveinteracting partner with the nucleic acid sequence, wherein thenucleotide has decreased binding affinity to the major groove bindingpartner.

In another aspect, the present disclosure provides compositionscomprising a compound as described herein. In some embodiments, thecomposition is a reaction mixture. In some embodiments, the compositionis a pharmaceutical composition. In some embodiments, the composition isa cell culture. In some embodiments, the composition further comprisesan RNA polymerase and a cDNA template. In some embodiments, thecomposition further comprises a nucleotide selected from the groupconsisting of adenosine, cytosine, guanosine, and uracil.

In a further aspect, the present disclosure provides methods of making apharmaceutical formulation comprising a physiologically active secretedprotein, comprising transfecting a first population of human cells withthe pharmaceutical nucleic acid made by the methods described herein,wherein the secreted protein is active upon a second population of humancells.

In some embodiments, the secreted protein is capable of interacting witha receptor on the surface of at least one cell present in the secondpopulation.

In some embodiments, the secreted protein is Granulocyte-ColonyStimulating Factor (G-CSF).

In some embodiments, the second population contains myeloblast cellsthat express the G-CSF receptor.

In certain embodiments, provided herein are combination therapeuticscontaining one or more modified nucleic acids containing translatableregions that encode for a protein or proteins that boost a mammaliansubject's immunity along with a protein that induces antibody-dependentcellular toxitity. For example, provided are therapeutics containing oneor more nucleic acids that encode trastuzumab and granulocyte-colonystimulating factor (G-CSF). In particular, such combination therapeuticsare useful in Her2+ breast cancer patients who develop inducedresistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy. 2(6):795-8 (2010)).

In one embodiment, it is intended that the compounds of the presentdisclosure are stable. It is further appreciated that certain featuresof the present disclosure, which are, for clarity, described in thecontext of separate embodiments, can also be provided in combination ina single embodiment. Conversely, various features of the presentdisclosure which are, for brevity, described in the context of a singleembodiment, can also be provided separately or in any suitablesubcombination.

Modified Nucleotides, Nucleosides and Polynucleotides of the Invention

Herein, in a nucleotide, nucleoside or polynucleotide (such as thenucleic acids of the invention, e.g., mRNA molecule), the terms“modification” or, as appropriate, “modified” refer to modification withrespect to A, G, U or C ribonucleotides. Generally, herein, these termsare not intended to refer to the ribonucleotide modifications innaturally occurring 5′-terminal mRNA cap moieties. In a polypeptide, theterm “modification” refers to a modification as compared to thecanonical set of 20 amino acids, moiety)

The modifications may be various distinct modifications. In someembodiments, where the nucleic acid is an mRNA, the coding region, theflanking regions and/or the terminal regions may contain one, two, ormore (optionally different) nucleoside or nucleotide modifications. Insome embodiments, a modified polynucleotide introduced to a cell mayexhibit reduced degradation in the cell, as compared to an unmodifiedpolynucleotide.

The polynucleotides can include any useful modification, such as to thesugar, the nucleobase, or the internucleoside linkage (e.g. to a linkingphosphate/to a phosphodiester linkage/to the phosphodiester backbone).For example, the major groove of a polynucleotide, or the major grooveface of a nucleobase may comprise one or more modifications. One or moreatoms of a pyrimidine nucleobase (e.g. on the major groove face) may bereplaced or substituted with optionally substituted amino, optionallysubstituted thiol, optionally substituted alkyl (e.g., methyl or ethyl),or halo (e.g., chloro or fluoro). In certain embodiments, modifications(e.g., one or more modifications) are present in each of the sugar andthe internucleoside linkage. Modifications according to the presentinvention may be modifications of ribonucleic acids (RNAs) todeoxyribonucleic acids (DNAs), e.g., the substitution of the 2′OH of theribofuranysyl ring to 2′H, threose nucleic acids (TNAs), glycol nucleicacids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs)or hybrids thereof). Additional modifications are described herein.

As described herein, the polynucleotides of the invention do notsubstantially induce an innate immune response of a cell into which thepolynucleotide (e.g., mRNA) is introduced. Features of an induced innateimmune response include 1) increased expression of pro-inflammatorycytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc, and/or3) termination or reduction in protein translation.

In certain embodiments, it may desirable for a modified nucleic acidmolecule introduced into the cell to be degraded intracellulary. Forexample, degradation of a modified nucleic acid molecule may bepreferable if precise timing of protein production is desired. Thus, insome embodiments, the invention provides a modified nucleic acidmolecule containing a degradation domain, which is capable of beingacted on in a directed manner within a cell. In another aspect, thepresent disclosure provides polynucleotides comprising a nucleoside ornucleotide that can disrupt the binding of a major groove interacting,e.g. binding, partner with the polynucleotide (e.g., where the modifiednucleotide has decreased binding affinity to major groove interactingpartner, as compared to an unmodified nucleotide).

The polynucleotides can optionally include other agents (e.g.,RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisenseRNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helixformation, aptamers, vectors, etc.). In some embodiments, thepolynucleotides may include one or more messenger RNAs (mRNAs) havingone or more modified nucleoside or nucleotides (i.e., modified mRNAmolecules). Details for these polynucleotides follow.

Polynucleotides

The polynucleotides of the invention includes a first region of linkednucleosides encoding a polypeptide of interest, a first flanking regionlocated at the 5′ terminus of the first region, and a second flankingregion located at the 3′ terminus of the first region.

In some embodiments, the polynucleotide (e.g., the first region, firstflanking region, or second flanking region) includes n number of linkednucleosides having Formula (Ia) or Formula (Ia-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Uis O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integer from0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

— is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R¹, R², R³, R⁴, and R⁵, ifpresent, is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, orabsent; wherein the combination of R³ with one or more of R^(1′),R^(1″), R^(2′), R^(2″), or R⁵ (e.g., the combination of R^(1′) and R³,the combination of R^(1″) and R³, the combination of R^(2′) and R³, thecombination of R^(2″) and R³, or the combination of R⁵ and R³) can jointogether to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein thecombination of R⁵ with one or more of R^(1′), R^(1″), R^(2′), or R^(2″)(e.g., the combination of R^(1′) and R⁵, the combination of R^(1″) andR⁵, the combination of R^(2′) and R⁵, or the combination of R^(2″) andR⁵) can join together to form optionally substituted alkylene oroptionally substituted heteroalkylene and, taken together with thecarbons to which they are attached, provide an optionally substitutedheterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl);and wherein the combination of R⁴ and one or more of R^(1′), R^(1″),R^(2′), R^(2″), R³, or R⁵ can join together to form optionallysubstituted alkylene or optionally substituted heteroalkylene and, takentogether with the carbons to which they are attached, provide anoptionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl);

each of m′ and m″ is, independently, an integer from 0 to 3 (e.g., from0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof), wherein the combination of B and R^(1′), the combination of Band R^(2′), the combination of B and R^(1″), or the combination of B andR^(2″) can, taken together with the carbons to which they are attached,optionally form a bicyclic group (e.g., a bicyclic heterocyclyl) orwherein the combination of B, R^(1″), and R³ or the combination of B,R^(2″), and R³ can optionally form a tricyclic or tetracyclic group(e.g., a tricyclic or tetracyclic heterocyclyl, such as in Formula(IIo)-(IIp) herein).

In some embodiments, the polynucleotide includes a modified ribose. Insome embodiments, the polynucleotide (e.g., the first region, the firstflanking region, or the second flanking region) includes n number oflinked nucleosides having Formula (Ia-2)-(Ia-5) or a pharmaceuticallyacceptable salt or stereoisomer thereof.

In some embodiments, the polynucleotide (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (Ib) or Formula (Ib-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

— is a single bond or absent;

each of R¹, R^(3′), R^(3″), and R⁴ is, independently, H, halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionallysubstituted hydroxyalkoxy, optionally substituted amino, azido,optionally substituted aryl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, or absent; and wherein the combination of R¹ and R^(3′) orthe combination of R¹ and R^(3″) can be taken together to formoptionally substituted alkylene or optionally substituted heteroalkylene(e.g., to produce a locked nucleic acid);

each R⁵ is, independently, H, halo, hydroxy, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, or absent;

each of Y¹, Y², and Y³ is, independently, O, S, Se, NR^(N1)—, optionallysubstituted alkylene, or optionally substituted heteroalkylene, whereinR^(N1) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, or optionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted alkoxyalkoxy, or optionally substituted amino;

n is an integer from 1 to 100,000; and

B is a nucleobase.

In some embodiments, the polynucleotide (e.g., the first region, firstflanking region, or second flanking region) includes n number of linkednucleosides having Formula (Ic):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

— is a single bond or absent;

each of B¹, B², and B³ is, independently, a nucleobase (e.g., a purine,a pyrimidine, or derivatives thereof, as described herein), H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl, wherein one and only one of B¹, B²,and B³ is a nucleobase;

each of R^(b1), R^(b2), R^(b3), R³, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

wherein the ring including U can include one or more double bonds.

In particular embodiments, the ring including U does not have a doublebond between U-CB³R^(b3) or between CB³R^(b3)—C^(B2)R^(b2).

In some embodiments, the polynucleotide (e.g., the first region, firstflanking region, or second flanking region) includes n number of linkednucleosides having Formula (Id):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Uis O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integer from0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

each R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, optionally substituted alkylene (e.g.,methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the polynucleotide (e.g., the first region, firstflanking region, or second flanking region) includes n number of linkednucleosides having Formula (Ie):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein each of U′ and U″ is, independently, O, S, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(u) is,independently, H, halo, or optionally substituted alkyl;

each R⁶ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each Y^(5′) is, independently, O, S, optionally substituted alkylene(e.g., methylene or ethylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the polynucleotide (e.g., the first region, firstflanking region, or second flanking region) includes n number of linkednucleosides having Formula (If) or (If-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein each of U′ and U″ is, independently, O, S, N,N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl (e.g., U′ is Oand U″ is N);

— is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R³, and R⁴ is, independently, H,halo, hydroxy, thiol, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,optionally substituted amino, azido, optionally substituted aryl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, or absent; and wherein thecombination of R^(1′) and R³, the combination of R^(1″) and R³, thecombination of R^(2′) and R³, or the combination of R^(2″) and R³ can betaken together to form optionally substituted alkylene or optionallysubstituted heteroalkylene (e.g., to produce a locked nucleic acid);each of m′ and m″ is, independently, an integer from 0 to 3 (e.g., from0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), the ring including U has one ortwo double bonds.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of R¹, R^(1′), and R^(1″),if present, is H. In further embodiments, each of R², R^(2′), andR^(2″), if present, is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl). In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′is C₁₋₆ alkyl.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of R², R^(2′), and R^(2″),if present, is H. In further embodiments, each of R¹, R^(1′), andR^(1″), if present, is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl). In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′is C₁₋₆ alkyl.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of R³, R⁴, and R⁵ is,independently, H, halo (e.g., fluoro), hydroxy, optionally substitutedalkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), oroptionally substituted alkoxyalkoxy. In particular embodiments, R³ is H,R⁴ is H, R⁵ is H, or R³, R⁴, and R⁵ are all H. In particularembodiments, R³ is C₁₋₆ alkyl, R⁴ is C₁₋₆ alkyl, R⁵ is C₁₋₆ alkyl, orR³, R⁴, and R⁵ are all C₁₋₆ alkyl. In particular embodiments, R³ and R⁴are both H, and R⁵ is C₁₋₆ alkyl.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R³ and R⁵ join together to formoptionally substituted alkylene or optionally substituted heteroalkyleneand, taken together with the carbons to which they are attached, providean optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl, such as trans-3′,4′ analogs, wherein R³ and R⁵join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R³ and one or more of R^(1′),R^(1″), R^(2′), R^(2″), or R⁵ join together to form optionallysubstituted alkylene or optionally substituted heteroalkylene and, takentogether with the carbons to which they are attached, provide anoptionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl, R³ and one or more of R^(1′), R^(1″), R^(2′),R^(2″), or R⁵ join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R⁵ and one or more of R^(1′),R^(1″), R^(2′), or R^(2″) join together to form optionally substitutedalkylene or optionally substituted heteroalkylene and, taken togetherwith the carbons to which they are attached, provide an optionallysubstituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclicheterocyclyl, R⁵ and one or more of R^(1′), R^(1″), R^(2′), or R^(2″)join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each Y² is, independently, O, S,or —NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl. In particular embodiments, Y² is NR^(N1)—,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl).

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(lip), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each Y³ is, independently, O orS.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R¹ is H; each R² is,independently, H, halo (e.g., fluoro), hydroxy, optionally substitutedalkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy(e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integerfrom 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ isC₁₋₆ alkyl); each Y² is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y³ is,independently, O or S (e.g., S). In further embodiments, R³ is H, halo(e.g., fluoro), hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy (e.g., methoxy or ethoxy), or optionally substitutedalkoxyalkoxy. In yet further embodiments, each Y¹ is, independently, Oor —NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl (e.g., wherein R^(N1) is H or optionallysubstituted alkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl,or n-propyl)); and each Y⁴ is, independently, H, hydroxy, thiol,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(lip), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each R¹ is, independently, H,halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g.,methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ isC₁₋₆ alkyl); R² is H; each Y² is, independently, O or —NR^(N1)—, whereinR^(N1) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, or optionally substituted aryl(e.g., wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y³ is,independently, O or S (e.g., S). In further embodiments, R³ is H, halo(e.g., fluoro), hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy (e.g., methoxy or ethoxy), or optionally substitutedalkoxyalkoxy. In yet further embodiments, each Y¹ is, independently, Oor —NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl (e.g., wherein R^(N1) is H or optionallysubstituted alkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl,or n-propyl)); and each Y⁴ is, independently, H, hydroxy, thiol,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in theβ-D (e.g., β-D-ribo) configuration.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in theα-L (e.g., α-L-ribo) configuration.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), one or more B is notpseudouridine (ψ) or 5-methyl-cytidine (m⁵C).

In some embodiments, about 10% to about 100% of n number of Bnucleobases is not ψ or m⁵C (e.g., from 10% to 20%, from 10% to 35%,from 10% to 50%, from 10% to 60%, from 10% to 75%, from 10% to 90%, from10% to 95%, from 10% to 98%, from 10% to 99%, from 20% to 35%, from 20%to 50%, from 20% to 60%, from 20% to 75%, from 20% to 90%, from 20% to95%, from 20% to 98%, from 20% to 99%, from 20% to 100%, from 50% to60%, from 50% to 75%, from 50% to 90%, from 50% to 95%, from 50% to 98%,from 50% to 99%, from 50% to 100%, from 75% to 90%, from 75% to 95%,from 75% to 98%, from 75% to 99%, and from 75% to 100% of n number of Bis not ψ or m⁵C). In some embodiments, B is not ψ or m⁵C.

In some embodiments of the polynucleotides (e.g., Formulas (Ia)-(Ia-5),(Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1),(IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), when B is an unmodifiednucleobase selected from cytosine, guanine, uracil and adenine, then atleast one of Y¹, Y², or Y³ is not O.

In some embodiments, the polynucleotide includes a modified ribose. Insome embodiments, the polynucleotide (e.g., the first region, the firstflanking region, or the second flanking region) includes n number oflinked nucleosides having Formula (IIa)-(IIc):

or a pharmaceutically acceptable salt or stereoisomer thereof. Inparticular embodiments, U is O or C(R^(U))_(nu), wherein nu is aninteger from 0 to 2 and each R^(u) is, independently, H, halo, oroptionally substituted alkyl (e.g., U is —CH₂— or —CH—). In otherembodiments, each of R¹, R², R³, R⁴, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or absent (e.g., each R¹ and R² is,independently H, halo, hydroxy, optionally substituted alkyl, oroptionally substituted alkoxy; each R³ and R⁴ is, independently, H oroptionally substituted alkyl; and R⁵ is H or hydroxy), and

is a single bond or double bond.

In particular embodiments, the polynucleotide (e.g., the first region,the first flanking region, or the second flanking region) includes nnumber of linked nucleosides having Formula (IIb-1)-(IIb-2):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In other embodiments, each of R¹ andR² is, independently, H, halo, hydroxy, thiol, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy). In particular embodiments, R² is hydroxy oroptionally substituted alkoxy (e.g., methoxy, ethoxy, or any describedherein).

In particular embodiments, the polynucleotide (e.g., the first region,the first flanking region, or the second flanking region) includes nnumber of linked nucleosides having Formula (IIc-1)-(IIc-4):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, U is O or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl (e.g., U is —CH₂— or —CH—). In some embodiments, eachof R¹, R², and R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy; and each R³ is, independently, H oroptionally substituted alkyl)). In particular embodiments, R² isoptionally substituted alkoxy (e.g., methoxy or ethoxy, or any describedherein). In particular embodiments, R¹ is optionally substituted alkyl,and R² is hydroxy. In other embodiments, R¹ is hydroxy, and R² isoptionally substituted alkyl. In further embodiments, R³ is optionallysubstituted alkyl.

In some embodiments, the polynucleotide includes an acyclic modifiedribose. In some embodiments, the polynucleotide (e.g., the first region,the first flanking region, or the second flanking region) includes nnumber of linked nucleosides having Formula (IId)-(IIf):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the polynucleotide includes an acyclic modifiedhexitol. In some embodiments, the polynucleotide (e.g., the firstregion, the first flanking region, or the second flanking region)includes n number of linked nucleosides having Formula (IIg)-(IIj):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the polynucleotide includes a sugar moiety having acontracted or an expanded ribose ring. In some embodiments, thepolynucleotide (e.g., the first region, the first flanking region, orthe second flanking region) includes n number of linked nucleosideshaving Formula (IIk)-(IIm):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of R^(1″), R^(1′), R^(2′), and R^(2″) is, independently, H, halo,hydroxy, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted alkenyloxy, optionally substituted alkynyloxy,optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy,or absent; and wherein the combination of R^(2′) and R³ or thecombination of R^(2″) and R³ can be taken together to form optionallysubstituted alkylene or optionally substituted heteroalkylene.

In some embodiments, the polynucleotide includes a locked modifiedribose. In some embodiments, the polynucleotide (e.g., the first region,the first flanking region, or the second flanking region) includes nnumber of linked nucleosides having Formula (IIn):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—) (e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the polynucleotide (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (IIn-1)-(II-n2):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—) (e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the polynucleotide includes a locked modifiedribose that forms a tetracyclic heterocyclyl. In some embodiments, thepolynucleotide (e.g., the first region, the first flanking region, orthe second flanking region) includes n number of linked nucleosideshaving Formula (IIo):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(12a), R^(12c), T^(1′), T^(1″), T^(2′), T^(2″), V¹, and V³ are asdescribed herein.

Any of the formulas for the polynucleotides can include one or morenucleobases described herein (e.g., Formulas (b1)-(b43)).

In one embodiment, the present invention provides methods of preparing apolynucleotide comprising at least one nucleotide that disrupts bindingof a major groove interacting partner with the nucleic acid, wherein thepolynucleotide comprises n number of nucleosides having Formula (Ia), asdefined herein:

the method comprising reacting a compound of Formula (IIIa), as definedherein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide comprising at least one nucleotide thatdisrupts binding of a major groove binding partner with thepolynucleotide sequence, the method comprising: reacting a compound ofFormula (IIIa), as defined herein, with a primer, a cDNA template, andan RNA polymerase.

In one embodiment, the present invention provides methods of preparing apolynucleotide comprising at least one nucleotide that disrupts bindingof a major groove interacting partner with the nucleic acid, wherein thepolynucleotide comprises n number of nucleosides having Formula (Ia-1),as defined herein:

the method comprising reacting a compound of Formula (IIIa-1), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide comprising at least one nucleotide (e.g.,modified mRNA molecule) that disrupts binding of a major groove bindingpartner with the polynucleotide sequence, the method comprising:reacting a compound of Formula (IIIa-1), as defined herein, with aprimer, a cDNA template, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing apolynucleotide comprising at least one nucleotide that disrupts bindingof a major groove interacting partner with the nucleic acid sequence,wherein the polynucleotide comprises n number of nucleosides havingFormula (Ia-2), as defined herein:

the method comprising reacting a compound of Formula (IIIa-2), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide comprising at least one nucleotide (e.g.,modified mRNA molecule) that disrupts binding of a major groove bindingpartner with the polynucleotide, the method comprising reacting acompound of Formula (IIIa-2), as defined herein, with a primer, a cDNAtemplate, and an RNA polymerase.

In some embodiments, the reaction may be repeated from 1 to about 7,000times. In any of the embodiments herein, B may be a nucleobase ofFormula (b1)-(b43).

The polynucleotides can optionally include 5′ and/or 3′ flankingregions, which are described herein.

Modified Nucleotides and Nucleosides

The present invention also includes the building blocks, e.g., modifiedribonucleosides, modified ribonucleotides, of the polynucleotides, e.g.,modified RNA (or mRNA) molecules. For example, these building blocks canbe useful for preparing the polynucleotides of the invention.

In some embodiments, the building block molecule has Formula (IIIa) or(IIIa-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinthe substituents are as described herein (e.g., for Formula (Ia) and(Ia-1)), and wherein when B is an unmodified nucleobase selected fromcytosine, guanine, uracil and adenine, then at least one of Y¹, Y², orY³ is not O.

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, has Formula (IVa)-(IVb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In particular embodiments, Formula (IVa) or (IVb) is combined with amodified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, Formula (IVa) or (IVb) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified guanine(e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified adenine(e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, has Formula (IVc)-(IVk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In particular embodiments, one of Formulas (IVc)-(IVk) is combined witha modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)).

In particular embodiments, one of Formulas (IVc)-(IVk) is combined witha modified cytosine (e.g., any one of formulas (b10)-(b14), (b24),(b25), and (b32)-(b36), such as formula (b10) or (b32)).

In particular embodiments, one of Formulas (IVc)-(IVk) is combined witha modified guanine (e.g., any one of formulas (b15)-(b17) and(b37)-(b40)).

In particular embodiments, one of Formulas (IVc)-(IVk) is combined witha modified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide has Formula (Va) or (Vb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide has Formula (IXa)-(IXd):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)).

In particular embodiments, one of Formulas (IXa)-(IXd) is combined witha modified guanine (e.g., any one of formulas (b15)-(b17) and(b37)-(b40)). In particular embodiments, one of Formulas (IXa)-(IXd) iscombined with a modified adenine (e.g., any one of formulas (b18)-(b20)and (b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide has Formula (IXe)-(IXg):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In particular embodiments, one of Formulas (IXe)-(IXg) is combined witha modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)).

In particular embodiments, one of Formulas (IXe)-(IXg) is combined witha modified cytosine (e.g., any one of formulas (b10)-(b14), (b24),(b25), and (b32)-(b36), such as formula (b10) or (b32)).

In particular embodiments, one of Formulas (IXe)-(IXg) is combined witha modified guanine (e.g., any one of formulas (b15)-(b17) and(b37)-(b40)).

In particular embodiments, one of Formulas (IXe)-(IXg) is combined witha modified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide has Formula (IXh)-(IXk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)).

In particular embodiments, one of Formulas (IXh)-(IXk) is combined witha modified guanine (e.g., any one of formulas (b15)-(b17) and(b37)-(b40)). In particular embodiments, one of Formulas (IXh)-(IXk) iscombined with a modified adenine (e.g., any one of formulas (b18)-(b20)and (b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide has Formula (IXl)-(IXr):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r1 and r2 is, independently, an integer from 0 to 5 (e.g., from 0to 3, from 1 to 3, or from 1 to 5) and B is as described herein (e.g.,any one of (b1)-(b43)).

In particular embodiments, one of Formulas (IXl)-(IXr) is combined witha modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)).

In particular embodiments, one of Formulas (IXl)-(IXr) is combined witha modified cytosine (e.g., any one of formulas (b10)-(b14), (b24),(b25), and (b32)-(b36), such as formula (b10) or (b32)).

In particular embodiments, one of Formulas (IXl)-(IXr) is combined witha modified guanine (e.g., any one of formulas (b15)-(b17) and(b37)-(b40)). In particular embodiments, one of Formulas (IXl)-(IXr) iscombined with a modified adenine (e.g., any one of formulas (b18)-(b20)and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide can be selected from the groupconsisting of:

pharmaceutically acceptable salt or stereoisomer thereof, wherein each ris, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide can be selected from the groupconsisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5) and s1 is as described herein.

In some embodiments, the building block molecule, which may beincorporated into a nucleic acid (e.g., RNA, mRNA, polynucleotide), is amodified uridine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide is a modified cytidine (e.g.,selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)). For example, the building block molecule, which may beincorporated into a polynucleotide can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide is a modified adenosine (e.g.,selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, is a modified guanosine (e.g.,selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the major groove chemical modification can includereplacement of C group at C-5 of the ring (e.g., for a pyrimidinenucleoside, such as cytosine or uracil) with N (e.g., replacement ofthe >CH group at C-5 with >NR^(N1) group, wherein R^(N1) is H oroptionally substituted alkyl). For example, the building block molecule,which may be incorporated into a polynucleotide can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In another embodiment, the major groove chemical modification caninclude replacement of the hydrogen at C-5 of cytosine with halo (e.g.,Br, Cl, F, or I) or optionally substituted alkyl (e.g., methyl). Forexample, the building block molecule, which may be incorporated into apolynucleotide can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In yet a further embodiment, the major groove chemical modification caninclude a fused ring that is formed by the NH₂ at the C-4 position andthe carbon atom at the C-5 position. For example, the building blockmolecule, which may be incorporated into a polynucleotide can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which may be incorporated into a polynucleotide (e.g., RNAor mRNA, as described herein), can be modified on the sugar of theribonucleic acid. For example, the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different substituents. Exemplarysubstitutions at the 2′-position include, but are not limited to, H,halo, optionally substituted C₁₋₆ alkyl; optionally substituted C₁₋₆alkoxy; optionally substituted C₆₋₁₀ aryloxy; optionally substitutedC₃₋₈cycloalkyl; optionally substituted C₃₋₈ cycloalkoxy; optionallysubstituted C₆₋₁₀ aryloxy; optionally substituted C₆₋₁₀ aryl-C₁₋₆alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy; a sugar (e.g.,ribose, pentose, or any described herein); a polyethyleneglycol (PEG),—O(CH₂CH₂O)_(n)CH₂CH₂OR′, where R is H or optionally substituted alkyl,and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16,from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to20); “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connectedby a C₁₋₆ alkylene or C₁₋₆ heteroalkylene bridge to the 4′-carbon of thesame ribose sugar, where exemplary bridges included methylene,propylene, ether, or amino bridges; aminoalkyl, as defined herein;aminoalkoxy, as defined herein; amino as defined herein; and amino acid,as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide moleculecan include nucleotides containing, e.g., arabinose, as the sugar.

Modifications on the Nucleobase

The present disclosure provides for modified nucleosides andnucleotides. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. In some embodiments, thenucleosides and nucleotides described herein are generally chemicallymodified on the major groove face. Exemplary non-limiting modificationsinclude an amino group, a thiol group, an alkyl group, a halo group, orany described herein. The modified nucleotides may by synthesized by anyuseful method, as described herein (e.g., chemically, enzymatically, orrecombinantly to include one or more modified or non-naturalnucleosides).

The modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil.

The modified nucleosides and nucleotides can include a modifiednucleobase. Examples of nucleobases found in RNA include, but are notlimited to, adenine, guanine, cytosine, and uracil. Examples ofnucleobase found in DNA include, but are not limited to, adenine,guanine, cytosine, and thymine. These nucleobases can be modified orwholly replaced to provide polynucleotide molecules having enhancedproperties, e.g., resistance to nucleases, stability, and theseproperties may manifest through disruption of the binding of a majorgroove binding partner. For example, the nucleosides and nucleotidesdescribed can be chemically modified on the major groove face. In someembodiments, the major groove chemical modifications can include anamino group, a thiol group, an alkyl group, or a halo group.

Table 1 below identifies the chemical faces of each canonicalnucleotide. Circles identify the atoms comprising the respectivechemical regions.

TABLE 1 Major Groove Minor Groove Face Face Pyrimdines Cytidine:

Uridine:

Purines Adenosine:

Guanosine:

Watson-Crick Base-pairing Face Pyrimidines Cytidine:

Uridine:

Purines Adenosine:

Guanosine:

In some embodiments, B is a modified uracil. Exemplary modified uracilsinclude those having Formula (b1)-(b5):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) or the combination of T^(2′) and T^(2″) join together (e.g., asin T²) to form O (oxo), S (thio), or Se (seleno);

each of V¹ and V² is, independently, O, S, N(R^(Vb))_(nv), orC(R^(Vb))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vb) is,independently, H, halo, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g.,trifluoroacetyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, or optionally substituted alkoxycarbonylalkoxy(e.g., optionally substituted with any substituent described herein,such as those selected from (1)-(21) for alkyl);

R¹⁰ is H, halo, optionally substituted amino acid, hydroxy, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aminoalkyl, optionallysubstituted hydroxyalkyl, optionally substituted hydroxyalkenyl,optionally substituted hydroxyalkynyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl;

R¹¹ is H or optionally substituted alkyl;

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, or optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy), optionally substituted carboxyalkoxy,optionally substituted carboxyaminoalkyl, or optionally substitutedcarbamoylalkyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl.

Other exemplary modified uracils include those having Formula (b6)-(b9):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) join together (e.g., as in T¹) or the combination of T^(2′) andT^(2″) join together (e.g., as in T²) to form O (oxo), S (thio), or Se(seleno), or each T¹ and T² is, independently, O (oxo), S (thio), or Se(seleno);

each of W¹ and W² is, independently, N(R^(Wa))_(nw) or C(R^(Wa))_(nw),wherein nw is an integer from 0 to 2 and each R^(Wa) is, independently,H, optionally substituted alkyl, or optionally substituted alkoxy;

each V³ is, independently, O, S, N(R^(Va))_(nv), or C(R^(Va))_(nv),wherein nv is an integer from 0 to 2 and each R^(Va) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), and wherein R^(Va) and R^(12c)taken together with the carbon atoms to which they are attached can formoptionally substituted cycloalkyl, optionally substituted aryl, oroptionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy and/or an O-protecting group), optionallysubstituted carboxyalkoxy, optionally substituted carboxyaminoalkyl,optionally substituted carbamoylalkyl, or absent;

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted alkaryl, optionally substitutedheterocyclyl, optionally substituted alkheterocyclyl, optionallysubstituted amino acid, optionally substituted alkoxycarbonylacyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedalkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,optionally substituted alkoxycarbonylalkynyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl,

wherein the combination of R^(12b) and T^(1′) or the combination ofR^(12b) and R^(12c) can join together to form optionally substitutedheterocyclyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl.

Further exemplary modified uracils include those having Formula(b28)-(b31):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T¹ and T² is, independently, O (oxo), S (thio), or Se (seleno);

each R^(Vb′) and R^(Vb″) is, independently, H, halo, optionallysubstituted amino acid, optionally substituted alkyl, optionallysubstituted haloalkyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl) (e.g., R^(Vb′) is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted aminoalkyl, e.g., substituted with an N-protecting group,such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl);

R^(12a) is H, optionally substituted alkyl, optionally substitutedcarboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, or optionally substituted aminoalkynyl; and

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substituted aminoalkynyl(e.g., e.g., substituted with an N-protecting group, such as anydescribed herein, e.g., trifluoroacetyl, or sulfoalkyl), optionallysubstituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl.

In particular embodiments, T¹ is O (oxo), and T² is S (thio) or Se(seleno). In other embodiments, T¹ is S (thio), and T² is O (oxo) or Se(seleno). In some embodiments, R^(Vb′) is H, optionally substitutedalkyl, or optionally substituted alkoxy.

In other embodiments, each R^(12a) and R^(12b) is, independently, H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted hydroxyalkyl. Inparticular embodiments, R^(12a) is H. In other embodiments, both R^(12a)and R^(12b) are H.

In some embodiments, each R^(Vb′) of R^(12b) is, independently,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g., trifluoroacetyl,or sulfoalkyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or optionally substituted acylaminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl). In some embodiments, the amino and/oralkyl of the optionally substituted aminoalkyl is substituted with oneor more of optionally substituted alkyl, optionally substituted alkenyl,optionally substituted sulfoalkyl, optionally substituted carboxy (e.g.,substituted with an O-protecting group), optionally substituted hydroxy(e.g., substituted with an O-protecting group), optionally substitutedcarboxyalkyl (e.g., substituted with an O-protecting group), optionallysubstituted alkoxycarbonylalkyl (e.g., substituted with an O-protectinggroup), or N-protecting group. In some embodiments, optionallysubstituted aminoalkyl is substituted with an optionally substitutedsulfoalkyl or optionally substituted alkenyl. In particular embodiments,R^(12a) and R^(Vb″) are both H. In particular embodiments, T¹ is O(oxo), and T² is S (thio) or Se (seleno).

In some embodiments, R^(Vb′) is optionally substitutedalkoxycarbonylalkyl or optionally substituted carbamoylalkyl.

In particular embodiments, the optional substituent for R^(12a),R^(12b), R^(12c), or R^(Va) is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently,is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4,from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl); or anamino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B is a modified cytosine. Exemplary modifiedcytosines include compounds of Formula (b10)-(b14):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(3′) and T^(3″) is, independently, H, optionally substitutedalkyl, optionally substituted alkoxy, or optionally substitutedthioalkoxy, or the combination of T^(3′) and T^(3″) join together (e.g.,as in T³) to form O (oxo), S (thio), or Se (seleno);

each V⁴ is, independently, O, S, N(R^(Vc)))_(nv), or C(R^(Vc))_(nv),wherein nv is an integer from 0 to 2 and each Rye is, independently, H,halo, optionally substituted amino acid, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl), wherein the combination of R^(13b)and R^(Vc) can be taken together to form optionally substitutedheterocyclyl;

each V⁵ is, independently, N(R^(Vd))_(nv), or C(R^(Vd))_(nv), wherein nvis an integer from 0 to 2 and each R^(Vd) is, independently, H, halo,optionally substituted amino acid, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl) (e.g., V⁵ is —CH or N);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl; and

each of R⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

Further exemplary modified cytosines include those having Formula(b32)-(b35):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T¹ and T³ is, independently, O (oxo), S (thio), or Se (seleno);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl,alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl (e.g.,R¹⁵ is H, and R¹⁶ is H or optionally substituted alkyl).

In some embodiments, R¹⁵ is H, and R¹⁶ is H or optionally substitutedalkyl. In particular embodiments, R¹⁴ is H, acyl, or hydroxyalkyl. Insome embodiments, R¹⁴ is halo. In some embodiments, both R¹⁴ and R¹⁵ areH. In some embodiments, both R¹⁵ and R¹⁶ are H. In some embodiments,each of R¹⁴ and R¹⁵ and R¹⁶ is H. In further embodiments, each ofR^(13a) and R^(13b) is independently, H or optionally substituted alkyl.

Further non-limiting examples of modified cytosines include compounds ofFormula (b36):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each R^(13b) is, independently, H, optionally substituted acyl,optionally substituted acyloxyalkyl, optionally substituted alkyl, oroptionally substituted alkoxy, wherein the combination of R^(13b) andR^(14b) can be taken together to form optionally substitutedheterocyclyl;

each R^(14a) and R^(14b) is, independently, H, halo, hydroxy, thiol,optionally substituted acyl, optionally substituted amino acid,optionally substituted alkyl, optionally substituted haloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted hydroxyalkyl (e.g., substituted with anO-protecting group), optionally substituted hydroxyalkenyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted acyloxyalkyl,optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl,phosphoryl, optionally substituted aminoalkyl, or optionally substitutedcarboxyaminoalkyl), azido, optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl; and

each of R⁵ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

In particular embodiments, R^(14b) is an optionally substituted aminoacid (e.g., optionally substituted lysine). In some embodiments, R^(14a)is H.

In some embodiments, B is a modified guanine. Exemplary modifiedguanines include compounds of Formula (b15)-(b17):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

Each of T^(4′), T^(4″), T^(5′), T^(5″), T^(6′), and T^(6″) is,independently, H, optionally substituted alkyl, or optionallysubstituted alkoxy, and wherein the combination of T^(4′) and T^(4″)(e.g., as in T⁴) or the combination of T⁵ and T^(5″) (e.g., as in T⁵) orthe combination of T^(6′) and T^(6″) join together (e.g., as in T⁶) formO (oxo), S (thio), or Se (seleno);

each of V⁵ and V⁶ is, independently, O, S, N(R^(Vd))_(nv), orC(R^(Vd))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vd) is,independently, H, halo, thiol, optionally substituted amino acid, cyano,amidine, optionally substituted aminoalkyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), optionally substitutedthioalkoxy, or optionally substituted amino; and

each of R¹⁷, R¹⁸, R^(19a), R^(19b), R²¹, R²², R²³, and R²⁴ is,independently, H, halo, thiol, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted thioalkoxy, optionally substituted amino, or optionallysubstituted amino acid.

Exemplary modified guanosines include compounds of Formula (b37)-(b40):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(4′) is, independently, H, optionally substituted alkyl, oroptionally substituted alkoxy, and each T⁴ is, independently, O (oxo), S(thio), or Se (seleno);

each of R¹⁸, R^(19a), R^(19b), and R²¹ is, independently, H, halo,thiol, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted thioalkoxy,optionally substituted amino, or optionally substituted amino acid.

In some embodiments, R¹⁸ is H or optionally substituted alkyl. Infurther embodiments, T⁴ is oxo. In some embodiments, each of R^(19a) andR^(19b) is, independently, H or optionally substituted alkyl.

In some embodiments, B is a modified adenine. Exemplary modifiedadenines include compounds of Formula (b18)-(b20):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each V⁷ is, independently, O, S, N(R^(Ve))_(nv), or C(R^(Ve))_(nv),wherein nv is an integer from 0 to 2 and each Rye is, independently, H,halo, optionally substituted amino acid, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy (e.g., optionally substituted with anysubstituent described herein, such as those selected from (1)-(21) foralkyl);

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl);

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy, or optionallysubstituted amino;

each R²⁸ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, or optionally substituted alkynyl; and

each R²⁹ is, independently, H, optionally substituted acyl, optionallysubstituted amino acid, optionally substituted carbamoylalkyl,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted alkoxy, or optionallysubstituted amino.

Exemplary modified adenines include compounds of Formula (b41)-(b43):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl); and

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy, or optionallysubstituted amino.

In some embodiments, R^(26a) is H, and R^(26b) is optionally substitutedalkyl. In some embodiments, each of R^(26a) and R^(26b) is,independently, optionally substituted alkyl. In particular embodiments,R²⁷ is optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy. In other embodiments, R²⁵ isoptionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy.

In particular embodiments, the optional substituent for R^(26a),R^(26b), or R²⁹ is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B may have Formula (b21):

wherein X¹² is, independently, O, S, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene, xa is aninteger from 0 to 3, and R^(12a) and T² are as described herein.

In some embodiments, B may have Formula (b22):

wherein R^(10′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R¹¹, R^(12a), T¹, and T² are asdescribed herein.

In some embodiments, B may have Formula (b23):

wherein R¹⁰ is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substituted thienyl, or optionallysubstituted pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁰); and wherein R¹¹ (e.g., Hor any substituent described herein), R^(12a) (e.g., H or anysubstituent described herein), Ti (e.g., oxo or any substituentdescribed herein), and T² (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B may have Formula (b24):

wherein R^(14′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted alkaryl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R^(13a), R^(13b), R¹⁵, and T³ are asdescribed herein.

In some embodiments, B may have Formula (b25):

wherein R^(14′) is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substituted thienyl, or optionallysubstituted pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁴ or R^(14′)); and whereinR^(13a) (e.g., H or any substituent described herein), R^(13b) (e.g., Hor any substituent described herein), R¹⁵ (e.g., H or any substituentdescribed herein), and T³ (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B is a nucleobase selected from the groupconsisting of cytosine, guanine, adenine, and uracil. In someembodiments, B may be:

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo^(s)U), 5-carboxymethyl-uridine(cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine(chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s2U),5-aminomethyl-2-thio-uridine (nm⁵s2U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s2U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm^(S)U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τcm⁵s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s2U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine(f^(s)Cm), N4,N4,2′-O-trimethyl-cytidine (m⁴²Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m′ A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N-6-methyl-adenosine (ms²m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N-6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N-6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N-6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N-6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶²A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N-6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N-6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface. For example, such modifications include replacing hydrogen on C-5of uracil or cytosine with alkyl (e.g., methyl) or halo.

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

In some embodiments, the modified nucleotide is a compound of FormulaXI:

wherein:

denotes a single or a double bond;

— denotes an optional single bond;

U is O, S, —NR^(a)—, or —CR^(a)R^(b)— when

denotes a single bond, or U is —CR^(a)— when

denotes a double bond;

Z is H, C₁₋₁₂ alkyl, or C₆₋₂₀ aryl, or Z is absent when

denotes a double bond; and

Z can be —CR^(a)R^(b)— and form a bond with A;

A is H, OH, NHR wherein R=alkyl or aryl or phosphoryl, sulfate, —NH₂,N₃, azido, —SH, N an amino acid, or a peptide comprising 1 to 12 aminoacids;

D is H, OH, NHR wherein R=alkyl or aryl or phosphoryl, —NH₂, —SH, anamino acid, a peptide comprising 1 to 12 amino acids, or a group ofFormula XII:

or A and D together with the carbon atoms to which they are attachedform a 5-membered ring;

X is O or S;

each of Y¹ is independently selected from —OR^(a1), —NR^(a1)R^(b1), and—SR^(a1);

each of Y² and Y³ are independently selected from O, —CR^(a)R^(b)—,NR^(c), S or a linker comprising one or more atoms selected from thegroup consisting of C, O, N, and S;

n is 0, 1, 2, or 3;

m is 0, 1, 2 or 3;

B is nucleobase;

R^(a) and R^(b) are each independently H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₂₋₁₂ alkynyl, or C₆₋₂₀ aryl;

R^(c) is H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group;

R^(a1) and R^(b1) are each independently H or a counterion; and

—OR^(c1) is OH at a pH of about 1 or —OR^(c1) is O⁻ at physiological pH;

provided that the ring encompassing the variables A, B, D, U, Z, Y² andY³ cannot be ribose.

In some embodiments, B is a nucleobase selected from the groupconsisting of cytosine, guanine, adenine, and uracil.

In some embodiments, the nucleobase is a pyrimidine or derivativethereof.

In some embodiments, the modified nucleotides are a compound of FormulaXI-a:

In some embodiments, the modified nucleotides are a compound of FormulaXI-b:

In some embodiments, the modified nucleotides are a compound of FormulaXI-c1, XI-c2, or XI-c3:

In some embodiments, the modified nucleotides are a compound of FormulaXI:

wherein:

denotes a single or a double bond;

— denotes an optional single bond;

U is O, S, —NR^(a)—, or —CR^(a)R^(b)— when

denotes a single bond, or U is —CR^(a)— when

denotes a double bond;

Z is H, C₁₋₁₂ alkyl, or C₆₋₂₀ aryl, or Z is absent when

denotes a double bond; and

Z can be —CR^(a)R^(b)— and form a bond with A;

A is H, OH, sulfate, —NH₂, —SH, an amino acid, or a peptide comprising 1to 12 amino acids;

D is H, OH, —NH₂, —SH, an amino acid, a peptide comprising 1 to 12 aminoacids, or a group of Formula XII:

or A and D together with the carbon atoms to which they are attachedform a 5-membered ring;

X is O or S;

each of Y¹ is independently selected from —OR^(a1), —NR^(a1)R^(b1), and—SR^(a1);

each of Y² and Y³ are independently selected from O, —CR^(a)R^(b)—,NR^(c), S or a linker comprising one or more atoms selected from thegroup consisting of C, O, N, and S;

n is 0, 1, 2, or 3;

m is 0, 1, 2 or 3;

B is a nucleobase of Formula XIII:

wherein:

V is N or positively charged NR^(c);

R³ is NR^(c)R^(d), —OR^(a), or —SR^(a);

R⁴ is H or can optionally form a bond with Y³;

R⁵ is H, —NR^(c)R^(d), or —OR^(a);

R^(a) and R^(b) are each independently H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₂₋₁₂ alkynyl, or C₆₋₂₀ aryl;

R^(c) is H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group;

R^(a1) and R^(b1) are each independently H or a counterion; and

—OR^(c1) is OH at a pH of about 1 or —OR¹¹ is O at physiological pH.

In some embodiments, B is:

wherein R³ is —OH, —SH, or

In some embodiments, B is:

In some embodiments, B is:

In some embodiments, the modified nucleotides are a compound of FormulaI-d:

In some embodiments, the modified nucleotides are a compound selectedfrom the group consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the modified nucleotides are a compound selectedfrom the group consisting of:

or a pharmaceutically acceptable salt thereof.

Modifications on the Internucleoside Linkage

The modified nucleotides, which may be incorporated into apolynucleotide molecule, can be modified on the internucleoside linkage(e.g., phosphate backbone). Herein, in the context of the polynucleotidebackbone, the phrases “phosphate” and “phosphodiester” are usedinterchangeably. Backbone phosphate groups can be modified by replacingone or more of the oxygen atoms with a different substituent. Further,the modified nucleosides and nucleotides can include the wholesalereplacement of an unmodified phosphate moiety with anotherinternucleoside linkage as described herein. Examples of modifiedphosphate groups include, but are not limited to, phosphorothioate,phosphoroselenates, boranophosphates, boranophosphate esters, hydrogenphosphonates, phosphoramidates, phosphorodiamidates, alkyl or arylphosphonates, and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bemodified by the replacement of a linking oxygen with nitrogen (bridgedphosphoramidates), sulfur (bridged phosphorothioates), and carbon(bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. While not wishing to be bound by theory, phosphorothioatelinked polynucleotide molecules are expected to also reduce the innateimmune response through weaker binding/activation of cellular innateimmune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine,5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine),5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein below.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The polynucleotides of the invention can include a combination ofmodifications to the sugar, the nucleobase, and/or the internucleosidelinkage. These combinations can include any one or more modificationsdescribed herein. For examples, any of the nucleotides described hereinin Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), (IIa)-(IIp), (IIb-1),(IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)can be combined with any of the nucleobases described herein (e.g., inFormulas (b1)-(b43) or any other described herein).

Synthesis of Polynucleotide Molecules

The polynucleotide molecules for use in accordance with the inventionmay be prepared according to any useful technique, as described herein.The modified nucleosides and nucleotides used in the synthesis ofpolynucleotide molecules disclosed herein can be prepared from readilyavailable starting materials using the following general methods andprocedures. Where typical or preferred process conditions (e.g.,reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are provided, a skilled artisan would be able tooptimize and develop additional process conditions. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of polynucleotide molecules of the present invention caninvolve the protection and deprotection of various chemical groups. Theneed for protection and deprotection, and the selection of appropriateprotecting groups can be readily determined by one skilled in the art.The chemistry of protecting groups can be found, for example, in Greene,et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons,1991, which is incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified polynucleotides or nucleicacids (e.g., polynucleotides or modified mRNA molecules) can be carriedout by any of numerous methods known in the art. An example methodincludes fractional recrystallization using a “chiral resolving acid”which is an optically active, salt-forming organic acid. Suitableresolving agents for fractional recrystallization methods are, forexample, optically active acids, such as the D and L forms of tartaricacid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid,malic acid, lactic acid or the various optically active camphorsulfonicacids. Resolution of racemic mixtures can also be carried out by elutionon a column packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22 (1): 72-78, (1994); Fukuhara et al., Biochemistry, 1 (4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The polynucleotides of the invention may or may not be uniformlymodified along the entire length of the molecule. For example, one ormore or all types of nucleotide (e.g., purine or pyrimidine, or any oneor more or all of A, G, U, C) may or may not be uniformly modified in apolynucleotide of the invention, or in a given predetermined sequenceregion thereof. In some embodiments, all nucleotides X in apolynucleotide of the invention (or in a given sequence region thereof)are modified, wherein X may any one of nucleotides A, G, U, C, or anyone of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C,G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the polynucleotide. One of ordinary skill in theart will appreciate that the nucleotide analogs or other modification(s)may be located at any position(s) of a polynucleotide such that thefunction of the polynucleotide is not substantially decreased. Amodification may also be a 5′ or 3′ terminal modification. Thepolynucleotide may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e. any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%).

In some embodiments, the polynucleotide includes a modified pyrimidine(e.g., a modified uracil/uridine/U or modified cytosine/cytidine/C). Insome embodiments, the uracil or uridine (generally: U) in thepolynucleotide molecule may be replaced with from about 1% to about 100%of a modified uracil or modified uridine (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100% of a modified uracil or modifieduridine). The modified uracil or uridine can be replaced by a compoundhaving a single unique structure or by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures, asdescribed herein). In some embodiments, the cytosine or cytidine(generally: C) in the polynucleotide molecule may be replaced with fromabout 1% to about 100% of a modified cytosine or modified cytidine(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%,from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10%to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100% of a modified cytosine or modified cytidine). The modifiedcytosine or cytidine can be replaced by a compound having a singleunique structure or by a plurality of compounds having differentstructures (e.g., 2, 3, 4 or more unique structures, as describedherein).

In some embodiments, the present disclosure provides methods ofsynthesizing a polynucleotide (e.g., the first region, first flankingregion, or second flanking region) including n number of linkednucleosides having Formula (Ia-1):

comprising:

a) reacting a nucleotide of Formula (IV-1):

with a phosphoramidite compound of Formula (V-1):

wherein Y⁹ is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino,thiol, optionally substituted amino acid, or a peptide (e.g., includingfrom 2 to 12 amino acids); and each P¹, P², and P³ is, independently, asuitable protecting group; and

denotes a solid support;

to provide a polynucleotide of Formula (VI-1):

and

b) oxidizing or sulfurizing the polynucleotide of Formula (V) to yield apolynucleotide of Formula (VII-1):

and

c) removing the protecting groups to yield the polynucleotide of Formula(Ia).

In some embodiments, steps a) and b) are repeated from 1 to about 10,000times. In some embodiments, the methods further comprise a nucleotideselected from the group consisting of A, C, G and U adenosine, cytosine,guanosine, and uracil. In some embodiments, the nucleobase may be apyrimidine or derivative thereof. In some embodiments, thepolynucleotide is translatable.

Other components of polynucleotides are optional, and are beneficial insome embodiments. For example, a 5′ untranslated region (UTR) and/or a3′ UTR are provided, wherein either or both may independently containone or more different nucleotide modifications. In such embodiments,nucleotide modifications may also be present in the translatable region.Also provided are polynucleotides containing a Kozak sequence.

Combinations of Nucleotides

Further examples of modified nucleotides and modified nucleotidecombinations are provided below in Table 2. These combinations ofmodified nucleotides can be used to form the polynucleotides of theinvention. Unless otherwise noted, the modified nucleotides may becompletely substituted for the natural nucleotides of thepolynucleotides of the invention. As a non-limiting example, the naturalnucleotide uridine may be substituted with a modified nucleosidedescribed herein. In another non-limiting example, the naturalnucleotide uridine may be partially substituted (e.g., about 0.1%, 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modifiednucleoside disclosed herein.

TABLE 2 Modified Nucleotide Modified Nucleotide Combinationα-thio-cytidine α-thio-cytidine/5-iodo-uridineα-thio-cytidine/N1-methyl-pseudo-uridine α-thio-cytidine/α-thio-uridineα-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudo-uridine about50% of the cytosines are α-thio-cytidine pseudoisocytidinepseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines areN1-methyl-pseudouridine and about 50% of uridines are pseudouridinepseudoisocytidine/about 25% of uridines are N1-methyl-pseudouridine andabout 25% of uridines are pseudouridine (e.g., 25%N1-methyl-pseudouridine/75% pseudouridine) pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/ about50% of uridines are 2-thio-uridine N4-acetyl-cytidineN4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine

Certain modified nucleotides and nucleotide combinations have beenexplored by the current inventors. These findings are described in U.S.Provisional Application No. 61/404,413, filed on Oct. 1, 2010, entitledEngineered Nucleic Acids and Methods of Use Thereof, U.S. patentapplication Ser. No. 13/251,840, filed on Oct. 3, 2011, entitledModified Nucleotides, and Nucleic Acids, and Uses Thereof, nowabandoned, U.S. patent application Ser. No. 13/481,127, filed on May 25,2012, entitled Modified Nucleotides, and Nucleic Acids, and UsesThereof, International Patent Publication No WO2012045075, filed on Oct.3, 2011, entitled Modified Nucleosides, Nucleotides, And Nucleic Acids,and Uses Thereof, U.S. Patent Publication No US20120237975 filed on Oct.3, 2011, entitled Engineered Nucleic Acids and Method of Use Thereof,and International Patent Publication No WO2012045082, which areincorporated by reference in their entireties.

Further examples of modified nucleotide combinations are provided belowin Table 3. These combinations of modified nucleotides can be used toform the polynucleotides of the invention.

TABLE 3 Modified Nucleotide Modified Nucleotide Combination modifiedcytidine having one or more modified cytidine with (b10)/pseudouridinenucleobases of Formula (b10) modified cytidine with(b10)/N1-methyl-pseudouridine modified cytidine with(b10)/5-methoxy-uridine modified cytidine with (b10)/5-methyl-uridinemodified cytidine with (b10)/5-bromo-uridine modified cytidine with(b10)/2-thio-uridine about 50% of cytidine substituted with modifiedcytidine (b10)/about 50% of uridines are 2-thio-uridine modifiedcytidine having one or more modified cytidine with (b32)/pseudouridinenucleobases of Formula (b32) modified cytidine with(b32)/N1-methyl-pseudouridine modified cytidine with(b32)/5-methoxy-uridine modified cytidine with (b32)/5-methyl-uridinemodified cytidine with (b32)/5-bromo-uridine modified cytidine with(b32)/2-thio-uridine about 50% of cytidine substituted with modifiedcytidine (b32)/about 50% of uridines are 2-thio-uridine modified uridinehaving one or more modified uridine with (b1)/N4-acetyl-cytidinenucleobases of Formula (b1) modified uridine with (b1)/5-methyl-cytidinemodified uridine having one or more modified uridine with(b8)/N4-acetyl-cytidine nucleobases of Formula (b8) modified uridinewith (b8)/5-methyl-cytidine modified uridine having one or more modifieduridine with (b28)/N4-acetyl-cytidine nucleobases of Formula (b28)modified uridine with (b28)/5-methyl-cytidine modified uridine havingone or more modified uridine with (b29)/N4-acetyl-cytidine nucleobasesof Formula (b29) modified uridine with (b29)/5-methyl-cytidine modifieduridine having one or more modified uridine with(b30)/N4-acetyl-cytidine nucleobases of Formula (b30) modified uridinewith (b30)/5-methyl-cytidine

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), (b24), (b25), or (b32)-(b35) (e.g., atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100% of, e.g., a compound of Formula (b10) or (b32)).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9), (b21)-(b23), or (b28)-(b31) (e.g., atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100% of, e.g., a compound of Formula (b1), (b8), (b28), (b29), or(b30)).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), (b24), (b25), or (b32)-(b35) (e.g.Formula (b10) or (b32)), and at least 25% of the uracils are replaced bya compound of Formula (b1)-(b9), (b21)-(b23), or (b28)-(b31) (e.g.Formula (b1), (b8), (b28), (b29), or (b30)) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

Modifications Including Linker and a Payload

The nucleobase of the nucleotide can be covalently linked at anychemically appropriate position to a payload, e.g., detectable agent ortherapeutic agent. For example, the nucleobase can be deaza-adenosine ordeaza-guanosine and the linker can be attached at the C-7 or C-8positions of the deaza-adenosine or deaza-guanosine. In otherembodiments, the nucleobase can be cytosine or uracil and the linker canbe attached to the N-3 or C-5 positions of cytosine or uracil. Scheme 1below depicts an exemplary modified nucleotide wherein the nucleobase,adenine, is attached to a linker at the C-7 carbon of 7-deaza adenine.In addition, Scheme 1 depicts the modified nucleotide with the linkerand payload, e.g., a detectable agent, incorporated onto the 3′ end ofthe mRNA. Disulfide cleavage and 1,2-addition of the thiol group ontothe propargyl ester releases the detectable agent. The remainingstructure (depicted, for example, as pApC5Parg in Scheme 1) is theinhibitor. The rationale for the structure of the modified nucleotidesis that the tethered inhibitor sterically interferes with the ability ofthe polymerase to incorporate a second base. Thus, it is critical thatthe tether be long enough to affect this function and that the inhibitorbe in a stereochemical orientation that inhibits or prohibits second andfollow on nucleotides into the growing polynucleotide strand.

Linker

The term “linker” as used herein refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., detectable or therapeutic agent, at asecond end. The linker is of sufficient length as to not interfere withincorporation into a nucleic acid sequence.

Examples of chemical groups that can be incorporated into the linkerinclude, but are not limited to, an alkyl, alkene, an alkyne, an amido,an ether, a thioether, an or an ester group. The linker chain can alsocomprise part of a saturated, unsaturated or aromatic ring, includingpolycyclic and heteroaromatic rings wherein the heteroaromatic ring isan aryl group containing from one to four heteroatoms, N, O or S.Specific examples of linkers include, but are not limited to,unsaturated alkanes, polyethylene glycols, and dextran polymers.

For example, the linker can include ethylene or propylene glycolmonomeric units, e.g., diethylene glycol, dipropylene glycol,triethylene glycol, tripropylene glycol, tetraethylene glycol, ortetraethylene glycol. In some embodiments, the linker can include adivalent alkyl, alkenyl, and/or alkynyl moiety. The linker can includean ester, amide, or ether moiety.

Other examples include cleavable moieties within the linker, such as,for example, a disulfide bond (—S—S—) or an azo bond (—N═N—), which canbe cleaved using a reducing agent or photolysis. A cleavable bondincorporated into the linker and attached to a modified nucleotide, whencleaved, results in, for example, a short “scar” or chemicalmodification on the nucleotide. For example, after cleaving, theresulting scar on a nucleotide base, which formed part of the modifiednucleotide, and is incorporated into a polynucleotide strand, isunreactive and does not need to be chemically neutralized. Thisincreases the ease with which a subsequent nucleotide can beincorporated during sequencing of a nucleic acid polymer template. Forexample, conditions include the use of tris(2-carboxyethyl)phosphine(TCEP), dithiothreitol (DTT) and/or other reducing agents for cleavageof a disulfide bond. A selectively severable bond that includes an amidobond can be cleaved for example by the use of TCEP or other reducingagents, and/or photolysis. A selectively severable bond that includes anester bond can be cleaved for example by acidic or basic hydrolysis.

Payload

The methods and compositions described herein are useful for deliveringa payload to a biological target. The payload can be used, e.g., forlabeling (e.g., a detectable agent such as a fluorophore), or fortherapeutic purposes (e.g., a cytotoxin or other therapeutic agent).

Payload: Therapeutic Agents

In some embodiments the payload is a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat.No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499,5,846,545) and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, Samarium 153 and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

Payload:Detectable Agents

Examples of detectable substances include various organic smallmolecules, inorganic compounds, nanoparticles, enzymes or enzymesubstrates, fluorescent materials, luminescent materials, bioluminescentmaterials, chemiluminescent materials, radioactive materials, andcontrast agents. Such optically-detectable labels include for example,without limitation, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonicacid; acridine and derivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′ tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine. In some embodiments,the detectable label is a fluorescent dye, such as Cy5 and Cy3.

Examples luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin.

Examples of suitable radioactive material include ¹⁸F, ⁶⁷Ga, ^(81m)Kr,⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl, ¹²⁵I, ³⁵S, ¹⁴C, or ³H, ^(99m)Tc (e.g.,as pertechnetate (technetate (VII), TcO₄ ⁻) either directly orindirectly, or other radioisotope detectable by direct counting ofradioemission or by scintillation counting.

In addition, contrast agents, e.g., contrast agents for MRI or NMR, forX-ray CT, Raman imaging, optical coherence tomography, absorptionimaging, ultrasound imaging, or thermal imaging can be used. Exemplarycontrast agents include gold (e.g., gold nanoparticles), gadolinium(e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide(SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmallsuperparamagnetic iron oxide (USPIO)), manganese chelates (e.g.,Mn-DPDP), barium sulfate, iodinated contrast media (iohexyl),microbubbles, or perfluorocarbons can also be used.

In some embodiments, the detectable agent is a non-detectable pre-cursorthat becomes detectable upon activation. Examples include fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE (VisEn Medical)).

When the compounds are enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, theenzymatic label is detected by determination of conversion of anappropriate substrate to product.

In vitro assays in which these compositions can be used include enzymelinked immunosorbent assays (ELISAs), immunoprecipitations,immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA),and Western blot analysis.

Labels other than those described herein are contemplated by the presentdisclosure, including other optically-detectable labels. Labels can beattached to the modified nucleotide of the present disclosure at anyposition using standard chemistries such that the label can be removedfrom the incorporated base upon cleavage of the cleavable linker.

Payload: Cell Penetrating Payloads

In some embodiments, the modified nucleotides and modified nucleic acidscan also include a payload that can be a cell penetrating moiety oragent that enhances intracellular delivery of the compositions. Forexample, the compositions can include a cell-penetrating peptidesequence that facilitates delivery to the intracellular space, e.g.,HIV-derived TAT peptide, penetratins, transportans, or hCT derivedcell-penetrating peptides, see, e.g., Caron et al., (2001) Mol. Ther. 3(3):310-8; Langel, Cell-Penetrating Peptides: Processes and Applications(CRC Press, Boca Raton Fla. 2002); El-Andaloussi et al., (2005) CurrPharm Des. 11 (28):3597-611; and Deshayes et al., (2005) Cell Mol LifeSci. 62(16):1839-49. The compositions can also be formulated to includea cell penetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space.

Payload:Biological Targets

The modified nucleotides and modified nucleic acids described herein canbe used to deliver a payload to any biological target for which aspecific ligand exists or can be generated. The ligand can bind to thebiological target either covalently or non-covalently.

Exemplary biological targets include biopolymers, e.g., antibodies,nucleic acids such as RNA and DNA, proteins, enzymes; exemplary proteinsinclude enzymes, receptors, and ion channels. In some embodiments thetarget is a tissue- or cell-type specific marker, e.g., a protein thatis expressed specifically on a selected tissue or cell type. In someembodiments, the target is a receptor, such as, but not limited to,plasma membrane receptors and nuclear receptors; more specific examplesinclude G-protein-coupled receptors, cell pore proteins, transporterproteins, surface-expressed antibodies, HLA proteins, MHC proteins andgrowth factor receptors.

Synthesis of Modified Nucleotides

The modified nucleosides and nucleotides disclosed herein can beprepared from readily available starting materials using the followinggeneral methods and procedures. It is understood that where typical orpreferred process conditions (i.e., reaction temperatures, times, moleratios of reactants, solvents, pressures, etc.) are given; other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of modified nucleosides and nucleotides can involve theprotection and deprotection of various chemical groups. The need forprotection and deprotection, and the selection of appropriate protectinggroups can be readily determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Greene, etal., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons,1991, which is incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleosides and nucleotidescan be carried out by any of numerous methods known in the art. Anexample method includes fractional recrystallization using a “chiralresolving acid” which is an optically active, salt-forming organic acid.Suitable resolving agents for fractional recrystallization methods are,for example, optically active acids, such as the D and L forms oftartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelicacid, malic acid, lactic acid or the various optically activecamphorsulfonic acids. Resolution of racemic mixtures can also becarried out by elution on a column packed with an optically activeresolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elutionsolvent composition can be determined by one skilled in the art.

Exemplary syntheses of modified nucleotides, which are incorporated intoa polynucleotides, e.g., RNA or mRNA, are provided below in Scheme 2through Scheme 12. Scheme 2 provides a general method forphosphorylation of nucleosides, including modified nucleosides.

Various protecting groups may be used to control the reaction. Forexample, Scheme 3 provides the use of multiple protecting anddeprotecting steps to promote phosphorylation at the 5′ position of thesugar, rather than the 2′ and 3′ hydroxyl groups. Scheme 3

Modified nucleotides can be synthesized in any useful manner. Schemes 4,5, and 8 provide exemplary methods for synthesizing modified nucleotideshaving a modified purine nucleobase; and Schemes 6 and 7 provideexemplary methods for synthesizing modified nucleotides having amodified pseudouridine or pseudoisocytidine, respectively.

Schemes 9 and 10 provide exemplary syntheses of modified nucleotides.Scheme 11 provides a non-limiting biocatalytic method for producingnucleotides.

Scheme 12 provides an exemplary synthesis of a modified uracil, wherethe N1 position on the major groove face is modified with R^(12b), asprovided elsewhere, and the 5′-position of ribose is phosphorylated. T¹,T², R^(12a), R^(12b), and r are as provided herein. This synthesis, aswell as optimized versions thereof, can be used to modify the majorgroove face of other pyrimidine nucleobases and purine nucleobases (seee.g., Formulas (b1)-(b43)) and/or to install one or more phosphategroups (e.g., at the 5′ position of the sugar). This alkylating reactioncan also be used to include one or more optionally substituted alkylgroup at any reactive group (e.g., amino group) in any nucleobasedescribed herein (e.g., the amino groups in the Watson-Crickbase-pairing face for cytosine, uracil, adenine, and guanine).

Modified nucleosides and nucleotides can also be prepared according tothe synthetic methods described in Ogata et al. Journal of OrganicChemistry 74:2585-2588, 2009; Purmal et al. Nucleic Acids Research22(1): 72-78, 1994; Fukuhara et al. Biochemistry 1(4): 563-568, 1962;and Xu et al. Tetrahedron 48(9): 1729-1740, 1992, each of which areincorporated by reference in their entirety.

Modified Nucleic Acids

The present disclosure provides nucleic acids (or polynucleotides),including RNAs such as mRNAs that contain one or more modifiednucleosides (termed “modified nucleic acids”) or nucleotides asdescribed herein, which have useful properties including the lack of asubstantial induction of the innate immune response of a cell into whichthe mRNA is introduced. Because these modified nucleic acids enhance theefficiency of protein production, intracellular retention of nucleicacids, and viability of contacted cells, as well as possess reducedimmunogenicity, these nucleic acids having these properties are alsotermed “enhanced nucleic acids” herein.

In addition, the present disclosure provides nucleic acids, which havedecreased binding affinity to a major groove interacting, e.g. binding,partner. For example, the nucleic acids are comprised of at least onenucleotide that has been chemically modified on the major groove face asdescribed herein.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that is or can be incorporated into an oligonucleotidechain. In this context, the term nucleic acid is used synonymously withpolynucleotide. Exemplary nucleic acids for use in accordance with thepresent disclosure include, but are not limited to, one or more of DNA,RNA including messenger mRNA (mRNA), hybrids thereof, RNAi-inducingagents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,catalytic DNA, RNAs that induce triple helix formation, aptamers,vectors, etc., described in detail herein.

Provided are modified nucleic acids containing a translatable region andone, two, or more than two different nucleoside modifications. In someembodiments, the modified nucleic acid exhibits reduced degradation in acell into which the nucleic acid is introduced, relative to acorresponding unmodified nucleic acid. Exemplary nucleic acids includeribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleicacids (TNAs), glycol nucleic acids (GNAs), or a hybrid thereof. Inpreferred embodiments, the modified nucleic acid includes messenger RNAs(mRNAs). As described herein, the nucleic acids of the presentdisclosure do not substantially induce an innate immune response of acell into which the mRNA is introduced.

In certain embodiments, it is desirable to intracellularly degrade amodified nucleic acid introduced into the cell, for example if precisetiming of protein production is desired. Thus, the present disclosureprovides a modified nucleic acid containing a degradation domain, whichis capable of being acted on in a directed manner within a cell.

Other components of nucleic acid are optional, and are beneficial insome embodiments. For example, a 5′ untranslated region (UTR) and/or a3′ UTR are provided, wherein either or both may independently containone or more different nucleoside modifications. In such embodiments,nucleoside modifications may also be present in the translatable region.Also provided are nucleic acids containing a Kozak sequence.

Additionally, provided are nucleic acids containing one or more intronicnucleotide sequences capable of being excised from the nucleic acid.

Further, provided are nucleic acids containing an internal ribosomeentry site (IRES). An IRES may act as the sole ribosome binding site, ormay serve as one of multiple ribosome binding sites of an mRNA. An mRNAcontaining more than one functional ribosome binding site may encodeseveral peptides or polypeptides that are translated independently bythe ribosomes (“multicistronic mRNA”). When nucleic acids are providedwith an IRES, further optionally provided is a second translatableregion. Examples of IRES sequences that can be used according to thepresent disclosure include without limitation, those from picornaviruses(e.g. FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (SIV) orcricket paralysis viruses (CrPV).

In another aspect, the present disclosure provides for nucleic acidsequences comprising at least two nucleotides, the nucleic acid sequencecomprising a nucleotide that disrupts binding of a major groove bindingpartner with the nucleic acid sequence, wherein the nucleotide hasdecreased binding affinity to the major groove binding partner.

In some embodiments, the nucleic acid is a compound of Formula XI-a:

wherein:

denotes an optional double bond;

— denotes an optional single bond;

U is O, S, —NR^(a)—, or —CR^(a)R^(b)— when

denotes a single bond, or U is —CR^(a)— when

denotes a double bond;

A is H, OH, phosphoryl, pyrophosphate, sulfate, —NH₂, —SH, an aminoacid, a peptide comprising 2 to 12 amino acids;

X is O or S;

each of Y¹ is independently selected from —OR^(a1), —NR^(a1)R^(b1), and—SR^(a1);

each of Y² and Y³ are independently selected from O, —CR^(a)R^(b)—,NR^(c), S or a linker comprising one or more atoms selected from thegroup consisting of C, O, N, and S;

R^(a) and R^(b) are each independently H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₂₋₁₂ alkynyl, or C₆₋₂₀ aryl;

R^(c) is H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group;

R^(a1) and R^(b1) are each independently H or a counterion;

—OR^(c1) is OH at a pH of about 1 or —OR^(c1) is O⁻ at physiological pH;and

B is nucleobase;

provided that the ring encompassing the variables A, B, D, U, Z, Y² andY³ cannot be ribose.

In some embodiments, B is a nucleobase of Formula XII-a, XII-b, orXII-c:

wherein:

denotes a single or double bond;

X is O or S;

U and W are each independently C or N;

V is O, S, C or N;

wherein when V is C then R¹ is H, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆alkynyl, halo, or —OR^(c), wherein C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀alkynyl are each optionally substituted with —OH, —NR^(a)R^(b), —SH,—C(O)R^(c), —C(O)OR^(c), —NHC(O)R^(c), or —NHC(O)OR^(c);

and wherein when V is O, S, or N then R¹ is absent;

R² is H, —OR^(c), —SR^(c), —NR^(a)R^(b), or halo;

or when V is C then R¹ and R² together with the carbon atoms to whichthey are attached can form a 5- or 6-membered ring optionallysubstituted with 1-4 substituents selected from halo, —OH, —SH,—NR^(a)R^(b), C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₁₋₂₀ alkoxy,or C₁₋₂₀ thioalkyl;

R³ is H or C₁₋₂₀ alkyl;

R⁴ is H or C₁₋₂₀ alkyl; wherein when

denotes a double bond then R⁴ is absent, or N—R⁴, taken together, formsa positively charged N substituted with C₁₋₂₀ alkyl;

R^(a) and R^(b) are each independently H, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl,C₂₋₂₀ alkynyl, or C₆₋₂₀ aryl; and

R^(c) is H, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group.

In some embodiments, B is a nucleobase of Formula XII-a1, XII-a2,XII-a3, XII-a4, or XII-a5:

In some embodiments, the nucleobase is a pyrimidine or derivativethereof.

In some embodiments, the nucleic acid contains a plurality ofstructurally unique compounds of Formula XI-a.

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula XI-a (e.g., at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula XI-a (e.g., at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines and 25% of theuracils are replaced by a compound of Formula XI-a (e.g., at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or about 100%).

In some embodiments, the nucleic acid is translatable.

In some embodiments, when the nucleic acid includes a nucleotidemodified with a linker and payload, for example, as described herein,the nucleotide modified with a linker and payload is on the 3′ end ofthe nucleic acid.

Major Groove Interacting Partners

As described herein, the phrase “major groove interacting partner”refers RNA recognition receptors that detect and respond to RNA ligandsthrough interactions, e.g. binding, with the major groove face of anucleotide or nucleic acid. As such, RNA ligands comprising modifiednucleotides or nucleic acids as described herein decrease interactionswith major groove binding partners, and therefore decrease an innateimmune response, or expression and secretion of pro-inflammatorycytokines, or both.

Example major groove interacting, e.g. binding, partners include, butare not limited to the following nucleases and helicases. Withinmembranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single-and double-stranded RNAs. Within the cytoplasm, members of thesuperfamily 2 class of DEX(D/H) helicases and ATPases can sense RNAs toinitiate antiviral responses. These helicases include the RIG-I(retinoic acid-inducible gene I) and MDA5 (melanomadifferentiation-associated gene 5). Other examples include laboratory ofgenetics and physiology 2 (LGP2), HIN-200 domain containing proteins, orHelicase-domain containing proteins.

Prevention or Reduction of Innate Cellular Immune Response

The term “innate immune response” includes a cellular response toexogenous single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death. Protein synthesis is alsoreduced during the innate cellular immune response. While it isadvantageous to eliminate the innate immune response in a cell which istriggered by introduction of exogenous nucleic acids, the presentdisclosure provides modified nucleic acids such as mRNAs thatsubstantially reduce the immune response, including interferonsignaling, without entirely eliminating such a response. In someembodiments, the immune response is reduced by 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as comparedto the immune response induced by a corresponding unmodified nucleicacid. Such a reduction can be measured by expression or activity levelof Type 1 interferons or the expression of interferon-regulated genessuch as the toll-like receptors (e.g., TLR7 and TLR8). Reduction or lackof induction of innate immune response can also be measured by decreasedcell death following one or more administrations of modified RNAs to acell population; e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%,or over 95% less than the cell death frequency observed with acorresponding unmodified nucleic acid. Moreover, cell death may affectfewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than0.01% of cells contacted with the modified nucleic acids.

In some embodiments, the modified nucleic acids, includingpolynucleotides and/or mRNA molecules are modified in such a way as tonot induce, or induce only minimally, an immune response by therecipient cell or organism. Such evasion or avoidance of an immuneresponse trigger or activation is a novel feature of the modifiedpolynucleotides of the present invention.

The present disclosure provides for the repeated introduction (e.g.,transfection) of modified nucleic acids into a target cell population,e.g., in vitro, ex vivo, or in vivo. The step of contacting the cellpopulation may be repeated one or more times (such as two, three, four,five or more than five times). In some embodiments, the step ofcontacting the cell population with the modified nucleic acids isrepeated a number of times sufficient such that a predeterminedefficiency of protein translation in the cell population is achieved.Given the reduced cytotoxicity of the target cell population provided bythe nucleic acid modifications, such repeated transfections areachievable in a diverse array of cell types in vitro and/or in vivo.

Polypeptide Variants

Provided are nucleic acids that encode variant polypeptides, which havea certain identity with a reference polypeptide sequence. The term“identity” as known in the art, refers to a relationship between thesequences of two or more peptides, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness between peptides, as determined by the number of matchesbetween strings of two or more amino acid residues. “Identity” measuresthe percent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant has the same or a similaractivity as the reference polypeptide. Alternatively, the variant has analtered activity (e.g., increased or decreased) relative to a referencepolypeptide. Generally, variants of a particular polynucleotide orpolypeptide of the present disclosure will have at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to that particularreference polynucleotide or polypeptide as determined by sequencealignment programs and parameters described herein and known to thoseskilled in the art.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of this present disclosure. For example, providedherein is any protein fragment of a reference protein (meaning apolypeptide sequence at least one amino acid residue shorter than areference polypeptide sequence but otherwise identical) 10, 20, 30, 40,50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length Inanother example, any protein that includes a stretch of about 20, about30, about 40, about 50, or about 100 amino acids which are about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, orabout 100% identical to any of the sequences described herein can beutilized in accordance with the present disclosure. In certainembodiments, a protein sequence to be utilized in accordance with thepresent disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or moremutations as shown in any of the sequences provided or referencedherein.

Polypeptide Libraries

Also provided are polynucleotide libraries containing nucleosidemodifications, wherein the polynucleotides individually contain a firstnucleic acid sequence encoding a polypeptide, such as an antibody,protein binding partner, scaffold protein, and other polypeptides knownin the art. Preferably, the polynucleotides are mRNA in a form suitablefor direct introduction into a target cell host, which in turnsynthesizes the encoded polypeptide.

In certain embodiments, multiple variants of a protein, each withdifferent amino acid modification(s), are produced and tested todetermine the best variant in terms of pharmacokinetics, stability,biocompatibility, and/or biological activity, or a biophysical propertysuch as expression level. Such a library may contain 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or over 10⁹ possible variants (includingsubstitutions, deletions of one or more residues, and insertion of oneor more residues).

Polypeptide-Nucleic Acid Complexes

Proper protein translation involves the physical aggregation of a numberof polypeptides and nucleic acids associated with the mRNA. Provided bythe present disclosure are protein-nucleic acid complexes, containing atranslatable mRNA having one or more nucleoside modifications (e.g., atleast two different nucleoside modifications) and one or morepolypeptides bound to the mRNA. Generally, the proteins are provided inan amount effective to prevent or reduce an innate immune response of acell into which the complex is introduced.

Untranslatable Modified Nucleic Acids

As described herein, provided are mRNAs having sequences that aresubstantially not translatable. Such mRNA is effective as a vaccine whenadministered to a mammalian subject.

Also provided are modified nucleic acids that contain one or morenoncoding regions. Such modified nucleic acids are generally nottranslated, but are capable of binding to and sequestering one or moretranslational machinery component such as a ribosomal protein or atransfer RNA (tRNA), thereby effectively reducing protein expression inthe cell. The modified nucleic acid may contain a small nucleolar RNA(sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) orPiwi-interacting RNA (piRNA).

Synthesis of Modified Nucleic Acids

Nucleic acids for use in accordance with the present disclosure may beprepared according to any available technique including, but not limitedto chemical synthesis, enzymatic synthesis, which is generally termed invitro transcription, enzymatic or chemical cleavage of a longerprecursor, etc. Methods of synthesizing RNAs are known in the art (see,e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach,Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn,P. (ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

Modified nucleic acids need not be uniformly modified along the entirelength of the molecule. Different nucleotide modifications and/orbackbone structures may exist at various positions in the nucleic acid.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of anucleic acid such that the function of the nucleic acid is notsubstantially decreased. A modification may also be a 5′ or 3′ terminalmodification. The nucleic acids may contain at a minimum one and atmaximum 100% modified nucleotides, or any intervening percentage, suchas at least 5% modified nucleotides, at least 10% modified nucleotides,at least 25% modified nucleotides, at least 50% modified nucleotides, atleast 80% modified nucleotides, or at least 90% modified nucleotides.For example, the nucleic acids may contain a modified pyrimidine such asuracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the nucleic acid is replaced with a modified uracil. Themodified uracil can be replaced by a compound having a single uniquestructure, or can be replaced by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures). In someembodiments, at least 5%, at least 10%, at least 25%, at least 50%, atleast 80%, at least 90% or 100% of the cytosine in the nucleic acid isreplaced with a modified cytosine. The modified cytosine can be replacedby a compound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

Generally, the shortest length of a modified mRNA of the presentdisclosure can be the length of an mRNA sequence that is sufficient toencode for a dipeptide. In another embodiment, the length of the mRNAsequence is sufficient to encode for a tripeptide. In anotherembodiment, the length of an mRNA sequence is sufficient to encode for atetrapeptide. In another embodiment, the length of an mRNA sequence issufficient to encode for a pentapeptide. In another embodiment, thelength of an mRNA sequence is sufficient to encode for a hexapeptide. Inanother embodiment, the length of an mRNA sequence is sufficient toencode for a heptapeptide. In another embodiment, the length of an mRNAsequence is sufficient to encode for an octapeptide. In anotherembodiment, the length of an mRNA sequence is sufficient to encode for anonapeptide. In another embodiment, the length of an mRNA sequence issufficient to encode for a decapeptide.

Examples of dipeptides that the modified nucleic acid sequences canencode for include, but are not limited to, carnosine and anserine.

In a further embodiment, the mRNA is greater than 30 nucleotides inlength. In another embodiment, the RNA molecule is greater than 35nucleotides in length. In another embodiment, the length is at least 40nucleotides. In another embodiment, the length is at least 45nucleotides. In another embodiment, the length is at least 55nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 80nucleotides. In another embodiment, the length is at least 90nucleotides. In another embodiment, the length is at least 100nucleotides. In another embodiment, the length is at least 120nucleotides. In another embodiment, the length is at least 140nucleotides. In another embodiment, the length is at least 160nucleotides. In another embodiment, the length is at least 180nucleotides. In another embodiment, the length is at least 200nucleotides. In another embodiment, the length is at least 250nucleotides. In another embodiment, the length is at least 300nucleotides. In another embodiment, the length is at least 350nucleotides. In another embodiment, the length is at least 400nucleotides. In another embodiment, the length is at least 450nucleotides. In another embodiment, the length is at least 500nucleotides. In another embodiment, the length is at least 600nucleotides. In another embodiment, the length is at least 700nucleotides. In another embodiment, the length is at least 800nucleotides. In another embodiment, the length is at least 900nucleotides. In another embodiment, the length is at least 1000nucleotides. In another embodiment, the length is at least 1100nucleotides. In another embodiment, the length is at least 1200nucleotides. In another embodiment, the length is at least 1300nucleotides. In another embodiment, the length is at least 1400nucleotides. In another embodiment, the length is at least 1500nucleotides. In another embodiment, the length is at least 1600nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 2000nucleotides. In another embodiment, the length is at least 2500nucleotides. In another embodiment, the length is at least 3000nucleotides. In another embodiment, the length is at least 4000nucleotides. In another embodiment, the length is at least 5000nucleotides, or greater than 5000 nucleotides.

For example, the modified nucleic acids described herein can be preparedusing methods that are known to those skilled in the art of nucleic acidsynthesis.

In some embodiments, the present disclosure provides methods, e.g.,enzymatic, of preparing a nucleic acid sequence comprising a nucleotidethat disrupts binding of a major groove binding partner with the nucleicacid sequence, wherein the nucleic acid sequence comprises a compound ofFormula XI-a:

wherein:

the nucleotide has decreased binding affinity to the major groovebinding partner;

denotes an optional double bond;

— denotes an optional single bond;

U is O, S, —NR^(a)—, or —CR^(a)R^(b)— when

denotes a single bond, or U is —CR^(a)— when

denotes a double bond;

A is H, OH, phosphoryl, pyrophosphate, sulfate, —NH₂, —SH, an aminoacid, a peptide comprising 2 to 12 amino acids;

X is O or S;

each of Y¹ is independently selected from —OR^(a1), —NR^(a1)R^(b1), and—SR^(a1);

each of Y² and Y³ are independently selected from O, —CR^(a)R^(b)—,NR^(c), S or a linker comprising one or more atoms selected from thegroup consisting of C, O, N, and S;

R^(a) and R^(b) are each independently H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₂₋₁₂ alkynyl, or C₆₋₂₀ aryl;

R^(c) is H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group;

R^(a1) and R^(b1) are each independently H or a counterion;

—OR^(c1) is OH at a pH of about 1 or —OR^(c1) is O⁻ at physiological pH;and B is nucleobase;

provided that the ring encompassing the variables A, B, D, U, Z, Y² andY³ cannot be ribose the method comprising reacting a compound of FormulaXIII:

with an RNA polymerase, and a cDNA template.

In some embodiments, the reaction is repeated from 1 to about 7,000times.

In some embodiments, B is a nucleobase of Formula XII-a, XII-b, orXII-c:

wherein:

denotes a single or double bond;

X is O or S;

U and W are each independently C or N;

V is O, S, C or N;

wherein when V is C then R¹ is H, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆alkynyl, halo, or —OR^(c), wherein C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀alkynyl are each optionally substituted with —OH, —NR^(a)R^(b), —SH,—C(O)R^(c), —C(O)OR^(c), —NHC(O)R^(c), or —NHC(O)OR^(c);

and wherein when V is O, S, or N then R¹ is absent;

R² is H, —OR^(c), —SR^(c), —NR^(a)R^(b), or halo;

or when V is C then R¹ and R² together with the carbon atoms to whichthey are attached can form a 5- or 6-membered ring optionallysubstituted with 1-4 substituents selected from halo, —OH, —SH,—NR^(a)R^(b), C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₁₋₂₀ alkoxy,or C₁₋₂₀ thioalkyl;

R³ is H or C₁₋₂₀ alkyl;

R⁴ is H or C₁₋₂₀ alkyl; wherein when

denotes a double bond then R⁴ is absent, or N—R⁴ taken together, forms apositively charged N substituted with C₁₋₂₀ alkyl;

R^(a) and R^(b) are each independently H, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl,C₂₋₂₀ alkynyl, or C₆₋₂₀ aryl; and

R^(c) is H, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group.

In some embodiments, B is a nucleobase of Formula XII-a1, XII-a2,XII-a3, XII-a4, or XII-a5:

In some embodiments, the methods further comprise a nucleotide selectedfrom the group consisting of adenosine, cytosine, guanosine, and uracil.

In some embodiments, the nucleobase is a pyrimidine or derivativethereof.

In another aspect, the present disclosure provides for methods ofamplifying a nucleic acid sequence comprising a nucleotide that disruptsbinding of a major groove binding partner with the nucleic acidsequence, the method comprising:

reacting a compound of Formula XI-d:

wherein:

the nucleotide has decreased binding affinity to the major groovebinding partner;

denotes a single or a double bond;

— denotes an optional single bond;

U is O, S, —NR^(a)—, or —CR^(a)R^(b)— when

denotes a single bond, or U is —CR^(a)— when

denotes a double bond;

Z is H, C₁₋₁₂ alkyl, or C₆₋₂₀ aryl, or Z is absent when

denotes a double bond; and

Z can be —CR^(a)R^(b)— and form a bond with A;

A is H, OH, phosphoryl, pyrophosphate, sulfate, —NH₂, —SH, an aminoacid, or a peptide comprising 1 to 12 amino acids;

X is O or S;

each of Y¹ is independently selected from —OR^(a1), —NR^(a1)R^(b1), and—SR^(a1);

each of Y² and Y³ are independently selected from O, —CR^(a)R^(b)—,NR^(c), S or a linker comprising one or more atoms selected from thegroup consisting of C, O, N, and S;

n is 0, 1, 2, or 3;

m is 0, 1, 2 or 3;

B is nucleobase;

R^(a) and R^(b) are each independently H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₂₋₁₂ alkynyl, or C₆₋₂₀ aryl;

R^(c) is H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group;

R^(a1) and R^(b1) are each independently H or a counterion; and

—OR^(c1) is OH at a pH of about 1 or —OR¹¹ is O at physiological pH;

provided that the ring encompassing the variables A, B, D, U, Z, Y² andY³ cannot be ribose with a primer, a cDNA template, and an RNApolymerase.

In some embodiments, B is a nucleobase of Formula XII-a, XII-b, orXII-c:

wherein:

denotes a single or double bond;

X is O or S;

U and W are each independently C or N;

V is O, S, C or N;

wherein when V is C then R¹ is H, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆alkynyl, halo, or —OR^(c), wherein C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀alkynyl are each optionally substituted with —OH, —NR^(a)R^(b), —SH,—C(O)R^(c), —C(O)OR^(c), —NHC(O)R^(c), or —NHC(O)OR^(c);

and wherein when V is O, S, or N then R¹ is absent;

R² is H, —OR^(c), —SR^(c), —NR^(a)R^(b), or halo;

or when V is C then R¹ and R² together with the carbon atoms to whichthey are attached can form a 5- or 6-membered ring optionallysubstituted with 1-4 substituents selected from halo, —OH, —SH,—NR^(a)R^(b), C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₁₋₂₀ alkoxy,or C₁₋₂₀ thioalkyl;

R³ is H or C₁₋₂₀ alkyl;

R⁴ is H or C₁₋₂₀ alkyl; wherein when

denotes a double bond then R⁴ is absent, or N—R⁴ taken together, forms apositively charged N substituted with C₁₋₂₀ alkyl;

R^(a) and R^(b) are each independently H, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl,C₂₋₂₀ alkynyl, or C₆₋₂₀ aryl; and

R^(c) is H, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group.

In some embodiments, B is a nucleobase of Formula XII-a1, XII-a2,XII-a3, XII-a4, or XII-a5:

In some embodiments, the methods further comprise a nucleotide selectedfrom the group consisting of adenosine, cytosine, guanosine, and uracil.

In some embodiments, the nucleobase is a pyrimidine or derivativethereof.

In some embodiments, the present disclosure provides for methods ofsynthesizing a pharmaceutical nucleic acid, comprising the steps of:

a) providing a complementary deoxyribonucleic acid (cDNA) that encodes apharmaceutical protein of interest;

selecting a nucleotide that is known to disrupt a binding of a majorgroove binding partner with a nucleic acid, wherein the nucleotide hasdecreased binding affinity to the major groove binding partner; and

c) contacting the provided cDNA and the selected nucleotide with an RNApolymerase, under conditions such that the pharmaceutical nucleic acidis synthesized.

In further embodiments, the pharmaceutical nucleic acid is a ribonucleicacid (RNA).

In still a further aspect of the present disclosure, the modifiednucleic acids can be prepared using solid phase synthesis methods.

In some embodiments, the present disclosure provides methods ofsynthesizing a nucleic acid comprising a compound of Formula XI-a:

wherein:

denotes an optional double bond;

— denotes an optional single bond;

U is O, S, —NR^(a)—, or —CR^(a)R^(b)— when

denotes a single bond, or U is —CR^(a)— when

denotes a double bond;

A is H, OH, phosphoryl, pyrophosphate, sulfate, —NH₂, —SH, an aminoacid, a peptide comprising 2 to 12 amino acids;

X is O or S;

each of Y¹ is independently selected from —OR^(a1), —NR^(a1)R^(b1), and—SR^(a);

each of Y² and Y³ are independently selected from O, —CR^(a)R^(b)—,NR^(c), S or a linker comprising one or more atoms selected from thegroup consisting of C, O, N, and S;

R^(a) and R^(b) are each independently H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₂₋₁₂ alkynyl, or C₆₋₂₀ aryl;

R^(c) is H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group;

R^(a1) and R^(b1) are each independently H or a counterion;

—OR^(c1) is OH at a pH of about 1 or —OR^(c1) is O⁻ at physiological pH;and

B is nucleobase;

provided that the ring encompassing the variables A, B, U, Z, Y² and Y³cannot be ribose;

comprising:

a) reacting a nucleotide of Formula XIII-a:

with a phosphoramidite compound of Formula XIII-b:

wherein:

denotes a solid support; and

P¹, P² and P³ are each independently suitable protecting groups;

to provide a nucleic acid of Formula XIV-a:

XIV-a and b) oxidizing or sulfurizing the nucleic acid of Formula XIV-ato yield a nucleic acid of Formula XIVb:

and c) removing the protecting groups to yield the nucleic acid ofFormula XI-a.

In some embodiments, the methods further comprise a nucleotide selectedfrom the group consisting of adenosine, cytosine, guanosine, and uracil.

In some embodiments, B is a nucleobase of Formula XIII:

wherein:

V is N or positively charged NR^(c);

R³ is NR^(c)R^(d), —OR^(a), or —SR^(a);

R⁴ is H or can optionally form a bond with Y³;

R⁵ is H, —NR^(c)R^(d), or —OR^(a);

R^(a) and R^(b) are each independently H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₂₋₁₂ alkynyl, or C₆₋₂₀ aryl; and

R^(c) is H, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, phenyl, benzyl, a polyethyleneglycol group, or an amino-polyethylene glycol group.

In some embodiments, steps a) and b) are repeated from 1 to about 10,000times.

Uses of Modified Nucleic Acids Therapeutic Agents

The modified nucleic acids described herein can be used as therapeuticagents. For example, a modified nucleic acid described herein can beadministered to an animal or subject, wherein the modified nucleic acidis translated in vivo to produce a therapeutic peptide in the animal orsubject. Accordingly, provided herein are compositions, methods, kits,and reagents for treatment or prevention of disease or conditions inhumans and other mammals. The active therapeutic agents of the presentdisclosure include modified nucleic acids, cells containing modifiednucleic acids or polypeptides translated from the modified nucleicacids, polypeptides translated from modified nucleic acids, cellscontacted with cells containing modified nucleic acids or polypeptidestranslated from the modified nucleic acids, tissues containing cellscontaining modified nucleic acids and organs containing tissuescontaining cells containing modified nucleic acids.

Provided are methods of inducing translation of a synthetic orrecombinant polynucleotide to produce a polypeptide in a cell populationusing the modified nucleic acids described herein. Such translation canbe in vivo, ex vivo, in culture, or in vitro. The cell population iscontacted with an effective amount of a composition containing a nucleicacid that has at least one nucleoside modification, and a translatableregion encoding the polypeptide. The population is contacted underconditions such that the nucleic acid is localized into one or morecells of the cell population and the recombinant polypeptide istranslated in the cell from the nucleic acid.

An effective amount of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell or improve therapeutic utility.

Aspects of the present disclosure are directed to methods of inducing invivo translation of a recombinant polypeptide in a mammalian subject inneed thereof. Therein, an effective amount of a composition containing anucleic acid that has at least one nucleoside modification and atranslatable region encoding the polypeptide is administered to thesubject using the delivery methods described herein. The nucleic acid isprovided in an amount and under other conditions such that the nucleicacid is localized into a cell or cells of the subject and therecombinant polypeptide is translated in the cell from the nucleic acid.The cell in which the nucleic acid is localized, or the tissue in whichthe cell is present, may be targeted with one or more than one rounds ofnucleic acid administration.

Other aspects of the present disclosure relate to transplantation ofcells containing modified nucleic acids to a mammalian subject.Administration of cells to mammalian subjects is known to those ofordinary skill in the art, such as local implantation (e.g., topical orsubcutaneous administration), organ delivery or systemic injection(e.g., intravenous injection or inhalation), as is the formulation ofcells in pharmaceutically acceptable carrier. Compositions containingmodified nucleic acids are formulated for administrationintramuscularly, transarterially, intraperitoneally, intravenously,intranasally, subcutaneously, endoscopically, transdermally, orintrathecally. In some embodiments, the composition is formulated forextended release.

The subject to whom the therapeutic agent is administered suffers fromor is at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

In certain embodiments, the administered modified nucleic acid directsproduction of one or more recombinant polypeptides that provide afunctional activity which is substantially absent in the cell in whichthe recombinant polypeptide is translated. For example, the missingfunctional activity may be enzymatic, structural, or gene regulatory innature.

In other embodiments, the administered modified nucleic acid directsproduction of one or more recombinant polypeptides that replace apolypeptide (or multiple polypeptides) that is substantially absent inthe cell in which the recombinant polypeptide is translated. Suchabsence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In other embodiments, the administeredmodified nucleic acid directs production of one or more recombinantpolypeptides to supplement the amount of polypeptide (or multiplepolypeptides) that is present in the cell in which the recombinantpolypeptide is translated. Alternatively, the recombinant polypeptidefunctions to antagonize the activity of an endogenous protein presentin, on the surface of, or secreted from the cell. Usually, the activityof the endogenous protein is deleterious to the subject, for example,due to mutation of the endogenous protein resulting in altered activityor localization. Additionally, the recombinant polypeptide antagonizes,directly or indirectly, the activity of a biological moiety present in,on the surface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, or asmall molecule toxin.

The recombinant proteins described herein are engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

As described herein, a useful feature of the modified nucleic acids ofthe present disclosure is the capacity to reduce, evade, avoid oreliminate the innate immune response of a cell to an exogenous nucleicacid. Provided are methods for performing the titration, reduction orelimination of the immune response in a cell or a population of cells.In some embodiments, the cell is contacted with a first composition thatcontains a first dose of a first exogenous nucleic acid including atranslatable region and at least one nucleoside modification, and thelevel of the innate immune response of the cell to the first exogenousnucleic acid is determined. Subsequently, the cell is contacted with asecond composition, which includes a second dose of the first exogenousnucleic acid, the second dose containing a lesser amount of the firstexogenous nucleic acid as compared to the first dose. Alternatively, thecell is contacted with a first dose of a second exogenous nucleic acid.The second exogenous nucleic acid may contain one or more modifiednucleosides, which may be the same or different from the first exogenousnucleic acid or, alternatively, the second exogenous nucleic acid maynot contain modified nucleosides. The steps of contacting the cell withthe first composition and/or the second composition may be repeated oneor more times. Additionally, efficiency of protein production (e.g.,protein translation) in the cell is optionally determined, and the cellmay be re-transfected with the first and/or second compositionrepeatedly until a target protein production efficiency is achieved.

Therapeutics for Diseases and Conditions

Provided are methods for treating or preventing a symptom of diseasescharacterized by missing or aberrant protein activity, by replacing themissing protein activity or overcoming the aberrant protein activity.Because of the rapid initiation of protein production followingintroduction of modified mRNAs, as compared to viral DNA vectors, thecompounds of the present disclosure are particularly advantageous intreating acute diseases such as sepsis, stroke, and myocardialinfarction. Moreover, the lack of transcriptional regulation of themodified mRNAs of the present disclosure is advantageous in thataccurate titration of protein production is achievable. Multiplediseases are characterized by missing (or substantially diminished suchthat proper protein function does not occur) protein activity. Suchproteins may not be present, are present in very low quantities or areessentially non-functional. The present disclosure provides a method fortreating such conditions or diseases in a subject by introducing nucleicacid or cell-based therapeutics containing the modified nucleic acidsprovided herein, wherein the modified nucleic acids encode for a proteinthat replaces the protein activity missing from the target cells of thesubject.

Diseases characterized by dysfunctional or aberrant protein activityinclude, but not limited to, cancer and proliferative diseases, geneticdiseases (e.g., cystic fibrosis), autoimmune diseases, diabetes,neurodegenerative diseases, cardiovascular diseases, and metabolicdiseases. The present disclosure provides a method for treating suchconditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the modified nucleic acids providedherein, wherein the modified nucleic acids encode for a protein thatantagonizes or otherwise overcomes the aberrant protein activity presentin the cell of the subject.

Specific examples of a dysfunctional protein are the missense ornonsense mutation variants of the cystic fibrosis transmembraneconductance regulator (CFTR) gene, which produce a dysfunctional ornonfunctional, respectively, protein variant of CFTR protein, whichcauses cystic fibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with a modified nucleic acidhaving a translatable region that encodes a functional CFTR polypeptide,under conditions such that an effective amount of the CTFR polypeptideis present in the cell. Preferred target cells are epithelial cells,such as the lung, and methods of administration are determined in viewof the target tissue; i.e., for lung delivery, the RNA molecules areformulated for administration by inhalation.

In another embodiment, the present disclosure provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with a modified mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721).

Methods of Cellular Nucleic Acid Delivery

Methods of the present disclosure enhance nucleic acid delivery into acell population, in vivo, ex vivo, or in culture. For example, a cellculture containing a plurality of host cells (e.g., eukaryotic cellssuch as yeast or mammalian cells) is contacted with a composition thatcontains an enhanced nucleic acid having at least one nucleosidemodification and, optionally, a translatable region. The compositionalso generally contains a transfection reagent or other compound thatincreases the efficiency of enhanced nucleic acid uptake into the hostcells. The enhanced nucleic acid exhibits enhanced retention in the cellpopulation, relative to a corresponding unmodified nucleic acid. Theretention of the enhanced nucleic acid is greater than the retention ofthe unmodified nucleic acid. In some embodiments, it is at least about50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greater than theretention of the unmodified nucleic acid. Such retention advantage maybe achieved by one round of transfection with the enhanced nucleic acid,or may be obtained following repeated rounds of transfection.

In some embodiments, the enhanced nucleic acid is delivered to a targetcell population with one or more additional nucleic acids. Such deliverymay be at the same time, or the enhanced nucleic acid is delivered priorto delivery of the one or more additional nucleic acids. The additionalone or more nucleic acids may be modified nucleic acids or unmodifiednucleic acids. It is understood that the initial presence of theenhanced nucleic acids does not substantially induce an innate immuneresponse of the cell population and, moreover, that the innate immuneresponse will not be activated by the later presence of the unmodifiednucleic acids. In this regard, the enhanced nucleic acid may not itselfcontain a translatable region, if the protein desired to be present inthe target cell population is translated from the unmodified nucleicacids.

Targeting Moieties

In embodiments of the present disclosure, modified nucleic acids areprovided to express a protein-binding partner or a receptor on thesurface of the cell, which functions to target the cell to a specifictissue space or to interact with a specific moiety, either in vivo or invitro. Suitable protein-binding partners include antibodies andfunctional fragments thereof, scaffold proteins, or peptides.Additionally, modified nucleic acids can be employed to direct thesynthesis and extracellular localization of lipids, carbohydrates, orother biological moieties.

Permanent Gene Expression Silencing

A method for epigenetically silencing gene expression in a mammaliansubject, comprising a nucleic acid where the translatable region encodesa polypeptide or polypeptides capable of directing sequence-specifichistone H3 methylation to initiate heterochromatin formation and reducegene transcription around specific genes for the purpose of silencingthe gene. For example, a gain-of-function mutation in the Janus Kinase 2gene is responsible for the family of Myeloproliferative Diseases.

Delivery of a Detectable or Therapeutic Agent to a Biological Target

The modified nucleosides, modified nucleotides, and modified nucleicacids described herein can be used in a number of different scenarios inwhich delivery of a substance (the “payload”) to a biological target isdesired, for example delivery of detectable substances for detection ofthe target, or delivery of a therapeutic agent. Detection methods caninclude both imaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

For example, the modified nucleosides, modified nucleotides, andmodified nucleic acids described herein can be used in reprogramminginduced pluripotent stem cells (iPS cells), which can then be used todirectly track cells that are transfected compared to total cells in thecluster. In another example, a drug that is attached to the modifiednucleic acid via a linker and is fluorescently labeled can be used totrack the drug in vivo, e.g. intracellularly. Other examples include theuse of a modified nucleic acid in reversible drug delivery into cells.

The modified nucleosides, modified nucleotides, and modified nucleicacids described herein can be used in intracellular targeting of apayload, e.g., detectable or therapeutic agent, to specific organelle.Exemplary intracellular targets can include the nuclear localization foradvanced mRNA processing, or a nuclear localization sequence (NLS)linked to the mRNA containing an inhibitor.

In addition, the modified nucleosides, modified nucleotides, andmodified nucleic acids described herein can be used to delivertherapeutic agents to cells or tissues, e.g., in living animals. Forexample, the modified nucleosides, modified nucleotides, and modifiednucleic acids described herein can be used to deliver highly polarchemotherapeutics agents to kill cancer cells. The modified nucleicacids attached to the therapeutic agent through a linker can facilitatemember permeation allowing the therapeutic agent to travel into a cellto reach an intracellular target.

In another example, the modified nucleosides, modified nucleotides, andmodified nucleic acids can be attached to a viral inhibitory peptide(VIP) through a cleavable linker. The cleavable linker will release theVIP and dye into the cell. In another example, the modified nucleosides,modified nucleotides, and modified nucleic acids can be attached throughthe linker to a ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins, causing massive fluid secretion from thelining of the small intestine, resulting in life-threatening diarrhea.

Pharmaceutical Compositions

The present disclosure provides proteins generated from modified mRNAs.Pharmaceutical compositions may optionally comprise one or moreadditional therapeutically active substances. In accordance with someembodiments, a method of administering pharmaceutical compositionscomprising a modified nucleic acide encoding one or more proteins to bedelivered to a subject in need thereof is provided. In some embodiments,compositions are administered to humans. For the purposes of the presentdisclosure, the phrase “active ingredient” generally refers to aprotein, protein encoding or protein-containing complex as describedherein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions is contemplated include, but are not limited to, humansand/or other primates; mammals, including commercially relevant mammalssuch as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;and/or birds, including commercially relevant birds such as chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” is discrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosurewill vary, depending upon the identity, size, and/or condition of thesubject treated and further depending upon the route by which thecomposition is to be administered. By way of example, the compositionmay comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipientmedium is incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thispresent disclosure.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan [Tween® 60],polyoxyethylene sorbitan monooleate [Tween® 80], sorbitan monopalmitate[Span® 40], sorbitan monostearate [Span® 60], sorbitan tristearate[Span® 65], glyceryl monooleate, sorbitan monooleate [Span® 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic®F 68, Poloxamer® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GlydantPlus®, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone™,Kathon™, and/or Euxyl®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as Cremophor®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. Solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present disclosurecontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid compositions to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions areconveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis present disclosure.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference).

Administration

The present disclosure provides methods comprising administeringproteins or complexes in accordance with the present disclosure to asubject in need thereof. Proteins or complexes, or pharmaceutical,imaging, diagnostic, or prophylactic compositions thereof, may beadministered to a subject using any amount and any route ofadministration effective for preventing, treating, diagnosing, orimaging a disease, disorder, and/or condition (e.g., a disease,disorder, and/or condition relating to working memory deficits). Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe disease, the particular composition, its mode of administration, itsmode of activity, and the like. Compositions in accordance with thepresent disclosure are typically formulated in dosage unit form for easeof administration and uniformity of dosage. It will be understood,however, that the total daily usage of the compositions of the presentdisclosure will be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective,prophylactically effective, or appropriate imaging dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

Proteins to be delivered and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof may be administered toanimals, such as mammals (e.g., humans, domesticated animals, cats,dogs, mice, rats, etc.). In some embodiments, pharmaceutical,prophylactic, diagnostic, or imaging compositions thereof areadministered to humans.

Proteins to be delivered and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof in accordance with thepresent disclosure may be administered by any route. In someembodiments, proteins and/or pharmaceutical, prophylactic, diagnostic,or imaging compositions thereof, are administered by one or more of avariety of routes, including oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, subcutaneous,intraventricular, transdermal, interdermal, rectal, intravaginal,intraperitoneal, topical (e.g. by powders, ointments, creams, gels,lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal,intratumoral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; as an oral spray, nasal spray, and/oraerosol, and/or through a portal vein catheter. In some embodiments,proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic,or imaging compositions thereof, are administered by systemicintravenous injection. In specific embodiments, proteins or complexesand/or pharmaceutical, prophylactic, diagnostic, or imaging compositionsthereof may be administered intravenously and/or orally. In specificembodiments, proteins or complexes, and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof, may be administered in away which allows the protein or complex to cross the blood-brainbarrier, vascular barrier, or other epithelial barrier.

However, the present disclosure encompasses the delivery of proteins orcomplexes, and/or pharmaceutical, prophylactic, diagnostic, or imagingcompositions thereof, by any appropriate route taking into considerationlikely advances in the sciences of drug delivery.

In general the most appropriate route of administration will depend upona variety of factors including the nature of the protein or complexcomprising proteins associated with at least one agent to be delivered(e.g., its stability in the environment of the gastrointestinal tract,bloodstream, etc.), the condition of the patient (e.g., whether thepatient is able to tolerate particular routes of administration), etc.The present disclosure encompasses the delivery of the pharmaceutical,prophylactic, diagnostic, or imaging compositions by any appropriateroute taking into consideration likely advances in the sciences of drugdelivery.

In certain embodiments, compositions in accordance with the presentdisclosure may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg toabout 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, fromabout 0.1 mg/kg to about mg/kg, or from about 1 mg/kg to about 25 mg/kg,of subject body weight per day, one or more times a day, to obtain thedesired therapeutic, diagnostic, prophylactic, or imaging effect. Thedesired dosage may be delivered three times a day, two times a day, oncea day, every other day, every third day, every week, every two weeks,every three weeks, or every four weeks. In certain embodiments, thedesired dosage may be delivered using multiple administrations (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations).

Proteins or complexes may be used in combination with one or more othertherapeutic, prophylactic, diagnostic, or imaging agents. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope of the presentdisclosure. Compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. In some embodiments, thepresent disclosure encompasses the delivery of pharmaceutical,prophylactic, diagnostic, or imaging compositions in combination withagents that improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination with be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually.

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a composition useful for treating cancer in accordance with thepresent disclosure may be administered concurrently with achemotherapeutic agent), or they may achieve different effects (e.g.,control of any adverse effects).

Kits

The present disclosure provides a variety of kits for convenientlyand/or effectively carrying out methods of the present disclosure.Typically kits will comprise sufficient amounts and/or numbers ofcomponents to allow a user to perform multiple treatments of asubject(s) and/or to perform multiple experiments.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and a nucleic acid modification, wherein the nucleic acid iscapable of evading or avoiding induction of an innate immune response ofa cell into which the first isolated nucleic acid is introduced, andpackaging and instructions.

In one aspect, the disclosure provides kits for protein production,comprising: a first isolated modified nucleic acid comprising atranslatable region, provided in an amount effective to produce adesired amount of a protein encoded by the translatable region whenintroduced into a target cell; a second nucleic acid comprising aninhibitory nucleic acid, provided in an amount effective tosubstantially inhibit the innate immune response of the cell; andpackaging and instructions.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and a nucleoside modification, wherein the nucleic acid exhibitsreduced degradation by a cellular nuclease, and packaging andinstructions.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and at least two different nucleoside modifications, wherein thenucleic acid exhibits reduced degradation by a cellular nuclease, andpackaging and instructions.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and at least one nucleoside modification,

wherein the nucleic acid exhibits reduced degradation by a cellularnuclease; a second nucleic acid comprising an inhibitory nucleic acid;and packaging and instructions.

In some embodiments, the first isolated nucleic acid comprises messengerRNA (mRNA). In some embodiments the mRNA comprises at least onenucleoside selected from the group consisting of pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine or any disclosedherein.

In some embodiments, the mRNA comprises at least one nucleoside selectedfrom the group consisting of 5-aza-cytidine, pseudoisocytidine,3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine,N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine or anydisclosed herein.

In some embodiments, the mRNA comprises at least one nucleoside selectedfrom the group consisting of 2-aminopurine, 2,6-diaminopurine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N-6-(cis-hydroxyisopentenyl)adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N-6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine or anydisclosed herein.

In some embodiments, the mRNA comprises at least one nucleoside selectedfrom the group consisting of inosine, 1-methyl-inosine, wyosine,wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine,6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine or anydisclosed herein.

In another aspect, the disclosure provides compositions for proteinproduction, comprising a first isolated nucleic acid comprising atranslatable region and a nucleoside modification, wherein the nucleicacid exhibits reduced degradation by a cellular nuclease, and amammalian cell suitable for translation of the translatable region ofthe first nucleic acid.

DEFINITIONS

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl.

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon

(IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosisfactor (TNF), such as TNF alpha and TNF beta, TNF gamma, TRAIL; G-CSF,GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide of the present invention may be considered biologicallyactive if even a portion of the polynucleotide is biologically active ormimics an activity considered biologically relevant.

Chemical terms: The following provides the definition of variouschemical terms from “acyl” to “thiol.”

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,trifluoroacetyl, propionyl, butanoyl and the like. Exemplaryunsubstituted acyl groups include from 1 to 7, from 1 to 11, or from 1to 21 carbons. In some embodiments, the alkyl group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “acylamino,” as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an aminogroup, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group (e.g.,haloalkyl) and R^(N1) is as defined herein). Exemplary unsubstitutedacylamino groups include from 1 to 41 carbons (e.g., from 1 to 7, from 1to 13, from 1 to 21, from 2 to 7, from 2 to 13, from 2 to 21, or from 2to 41 carbons). In some embodiments, the alkyl group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein, and/orthe amino group is —NH₂ or —NHR^(N1), wherein R^(N1) is, independently,OH, NO₂, NH₂, NR^(N22), SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, aryl,acyl (e.g., acetyl, trifluoroacetyl, or others described herein), oralkoxycarbonylalkyl, and each R^(N2) can be H, alkyl, or aryl.

The term “acylaminoalkyl,” as used herein, represents an acyl group, asdefined herein, attached to an amino group that is in turn attached tothe parent molecular group though an alkyl group, as defined herein(i.e., -alkyl-N(R^(N1))—C(O)—R, where R is H or an optionallysubstituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group (e.g., haloalkyl) andR^(N1) is as defined herein). Exemplary unsubstituted acylamino groupsinclude from 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to21, from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons).In some embodiments, the alkyl group is further substituted with 1, 2,3, or 4 substituents as described herein, and/or the amino group is —NH₂or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N22),SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, aryl, acyl (e.g., acetyl,trifluoroacetyl, or others described herein), or alkoxycarbonylalkyl,and each R^(N2) can be H, alkyl, or aryl.

The term “acyloxy,” as used herein, represents an acyl group, as definedherein, attached to the parent molecular group though an oxygen atom(i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groups includefrom 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein.

The term “acyloxyalkyl,” as used herein, represents an acyl group, asdefined herein, attached to an oxygen atom that in turn is attached tothe parent molecular group though an alkyl group (i.e., -alkyl-O—C(O)—R,where R is H or an optionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkylgroup). Exemplary unsubstituted acyloxyalkyl groups include from 1 to 21carbons (e.g., from 1 to 7 or from 1 to 11 carbons). In someembodiments, the alkyl group is, independently, further substituted with1, 2, 3, or 4 substituents as described herein.

The term “alkaryl,” as used herein, represents an aryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkaryl groups arefrom 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, suchas C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl).In some embodiments, the alkylene and the aryl each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein forthe respective groups. Other groups preceded by the prefix “alk-” aredefined in the same manner, where “alk” refers to a C₁₋₆ alkylene,unless otherwise noted, and the attached chemical structure is asdefined herein.

The term “alkcycloalkyl” represents a cycloalkyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein (e.g., an alkylene group of from 1 to 4, from 1to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, thealkylene and the cycloalkyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkenyloxy” represents a chemical substituent of formula —OR′,where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆ or C₂₋₁₀ alkenyl), unlessotherwise specified. Exemplary alkenyloxy groups include ethenyloxy,propenyloxy, and the like. In some embodiments, the alkenyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “alkheteroaryl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheteroaryl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Alkheteroaryl groups are a subset of alkheterocyclyl groups.

The term “alkheterocyclyl” represents a heterocyclyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheterocyclyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂ heterocyclyl). In some embodiments, thealkylene and the heterocyclyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkoxy” represents a chemical substituent of formula —OR′,where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆ or C₁₋₁₀ alkyl), unlessotherwise specified. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. Insome embodiments, the alkyl group can be further substituted with 1, 2,3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).

The term “alkoxyalkoxy” represents an alkoxy group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groupsinclude between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀alkoxy-C₁₋₁₀ alkoxy, orC₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments, the each alkoxy groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkoxyalkyl” represents an alkyl group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons,such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀alkoxy-C₁₋₂₀ alkyl). In some embodiments, the alkyl and the alkoxy eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy, asdefined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR′, where R is H or an optionallysubstituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkoxycarbonylacyl,” as used herein, represents an acyl group,as defined herein, that is substituted with an alkoxycarbonyl group, asdefined herein (e.g., —C(O)-alkyl-C(O)—OR′, where R is an optionallysubstituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonylacyl include from 3 to 41 carbons (e.g., from 3 to 10,from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, suchas C₁₋₆ alkoxycarbonyl-C₁₋₆ acyl, C₁₋₁₀ alkoxycarbonyl-C₁₋₁₀ acyl, orC₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ acyl). In some embodiments, each alkoxy andalkyl group is further independently substituted with 1, 2, 3, or 4substituents, as described herein (e.g., a hydroxy group) for eachgroup.

The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxygroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —O-alkyl-C(O)—OR′, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The term “alkoxycarbonylalkyl,” as used herein, represents an alkylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkyl-C(O)—OR′, where R is anoptionally substituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkyl include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl,C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkyl, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl).In some embodiments, each alkyl and alkoxy group is furtherindependently substituted with 1, 2, 3, or 4 substituents as describedherein (e.g., a hydroxy group).

The term “alkoxycarbonylalkenyl,” as used herein, represents an alkenylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkenyl-C(O)—OR′, where R is anoptionally substituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkenyl include from 4 to 41 carbons (e.g.,from 4 to 10, from 4 to 13, from 4 to 17, from 4 to 21, or from 4 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₂₋₆alkenyl, C₁₋₁₀alkoxycarbonyl-C₂₋₁₀ alkenyl, or C₁₋₂₀ alkoxycarbonyl-C₂₋₂₀ alkenyl). Insome embodiments, each alkyl, alkenyl, and alkoxy group is furtherindependently substituted with 1, 2, 3, or 4 substituents as describedherein (e.g., a hydroxy group).

The term “alkoxycarbonylalkynyl,” as used herein, represents an alkynylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkynyl-C(O)—OR′, where R is anoptionally substituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkynyl include from 4 to 41 carbons (e.g.,from 4 to 10, from 4 to 13, from 4 to 17, from 4 to 21, or from 4 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₂₋₆alkynyl, C₁₋₁₀alkoxycarbonyl-C₂₋₁₀ alkynyl, or C₁₋₂₀ alkoxycarbonyl-C₂₋₂₀ alkynyl). Insome embodiments, each alkyl, alkynyl, and alkoxy group is furtherindependently substituted with 1, 2, 3, or 4 substituents as describedherein (e.g., a hydroxy group).

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉heterocyclyl)oxy; (8)hydroxy, optionally substituted with an O-protecting group; (9) nitro;(10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇-spirocyclyl; (12)thioalkoxy; (13) thiol; (14) —CO₂R^(A′), optionally substituted with anO-protecting group and where R^(A′) is selected from the groupconsisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl(e.g., C₂₋₆alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R¹, wherein R^(H′) is selected from the group consisting of(a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from the groupconsisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl(e.g., C₂₋₆alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆ alk-C₆₋₁₀aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “alkynyloxy” represents a chemical substituent of formula —OR′,where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆ or C₂₋₁₀ alkynyl), unlessotherwise specified. Exemplary alkynyloxy groups include ethynyloxy,propynyloxy, and the like. In some embodiments, the alkynyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionallysubstituted with an O-protecting group, such as optionally substitutedarylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl(e.g., acetyl, trifluoroacetyl, or others described herein),alkoxycarbonylalkyl (e.g., optionally substituted with an O-protectinggroup, such as optionally substituted arylalkoxycarbonyl groups or anydescribed herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl(e.g., alkheteroaryl), wherein each of these recited R^(N1) groups canbe optionally substituted, as defined herein for each group; or twoR^(N1) combine to form a heterocyclyl or an N-protecting group, andwherein each R^(N2) is, independently, H, alkyl, or aryl. The aminogroups of the invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others describedherein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl) or aryl, andeach R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉heterocyclyl)oxy; (8) hydroxy; (9)nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇-spirocyclyl;(12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′) is selectedfrom the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e)C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂H₂O)_(s)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R¹, wherein R^(H′) is selected from the group consisting of(a1) hydrogen and (b1) C₁₋₆ alkyl, and R′ is selected from the groupconsisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl(e.g., C₂₋₆alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆ alk-C₆₋₁₀aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aminoalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy, and/or an N-protecting group).

The term “aminoalkenyl,” as used herein, represents an alkenyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkenyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy, and/or an N-protecting group).

The term “aminoalkynyl,” as used herein, represents an alkynyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkynyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy, and/or an N-protecting group).

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl;(10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g.,C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15)nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17)—(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, and R^(A′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkoxy,” as used herein, represents an alkaryl group, asdefined herein, attached to the parent molecular group through an oxygenatom. Exemplary unsubstituted arylalkoxy groups include from 7 to 30carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein

The term “arylalkoxycarbonyl,” as used herein, represents an arylalkoxygroup, as defined herein, attached to the parent molecular group througha carbonyl (e.g., —C(O)—O-alkyl-aryl). Exemplary unsubstitutedarylalkoxy groups include from 8 to 31 carbons (e.g., from 8 to 17 orfrom 8 to 21 carbons, such as C₆₋₁₀ aryl-C₁₋₆ alkoxy-carbonyl, C₆₋₁₀aryl-C₁₋₁₀ alkoxy-carbonyl, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy-carbonyl). Insome embodiments, the arylalkoxycarbonyl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified. In some embodiments, the aryl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloyl,” as used herein, represents an aryl group, as definedherein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The term “boranyl,” as used herein, represents —B(R^(B1))₃, where eachR^(B1) is, independently, selected from the group consisting of H andoptionally substituted alkyl. In some embodiments, the boranyl group canbe substituted with 1, 2, 3, or 4 substituents as defined herein foralkyl.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicstructure in which the rings, which may be aromatic or non-aromatic, areformed by carbon atoms. Carbocyclic structures include cycloalkyl,cycloalkenyl, and aryl groups.

The term “carbamoyl,” as used herein, represents —C(O)—N(R^(N1))₂, wherethe meaning of each R^(N1) is found in the definition of “amino”provided herein.

The term “carbamoylalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carbamoyl group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “carbamyl,” as used herein, refers to a carbamate group havingthe structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning ofeach R^(N1) is found in the definition of “amino” provided herein, and Ris alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents an acyl group having the structure—CHO.

The term “carboxy,” as used herein, means —CO₂H.

The term “carboxyalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkoxy group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein for the alkyl group, and the carboxy groupcan be optionally substituted with one or more O-protecting groups.

The term “carboxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein, and the carboxy group can be optionallysubstituted with one or more O-protecting groups.

The term “carboxyaminoalkyl,” as used herein, represents an aminoalkylgroup, as defined herein, substituted by a carboxy, as defined herein.The carboxy, alkyl, and amino each can be further substituted with 1, 2,3, or 4 substituent groups as described herein for the respective group(e.g., CO₂R^(A′), where R^(A′) is selected from the group consisting of(a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀aryl, e.g., carboxy, and/or an N-protecting group, and/or anO-protecting group).

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkoxy” represents a chemical substituent of formula—OR′, where R is a C₃₋₈cycloalkyl group, as defined herein, unlessotherwise specified. The cycloalkyl group can be further substitutedwith 1, 2, 3, or 4 substituent groups as described herein. Exemplaryunsubstituted cycloalkoxy groups are from 3 to 8 carbons. In someembodiment, the cycloalkyl group can be further substituted with 1, 2,3, or 4 substituent groups as described herein.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycle heptyl, andthe like. When the cycloalkyl group includes one carbon-carbon doublebond, the cycloalkyl group can be referred to as a “cycloalkenyl” group.Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, andthe like. The cycloalkyl groups of this invention can be optionallysubstituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl;(10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g.,C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15)nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17)—(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, and R^(A′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCCl₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which one or two of the constituent carbon atomshave each been replaced by nitrogen, oxygen, or sulfur. In someembodiments, the heteroalkylene group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkylene groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c, d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where E′ is selected from the group consisting of —N— and —CH—; F′ isselected from the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—,—CH═N—, —CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—,—O—, and —S—; and G′ is selected from the group consisting of —CH— and—N—. Any of the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “(heterocyclyl) imino,” as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group. In someembodiments, the hydroxy group can be substituted with 1, 2, 3, or 4substituent groups (e.g., O-protecting groups) as defined herein for analkyl.

The term “hydroxyalkenyl,” as used herein, represents an alkenyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by dihydroxypropenyl,hydroxyisopentenyl, and the like. In some embodiments, thehydroxyalkenyl group can be substituted with 1, 2, 3, or 4 substituentgroups (e.g., O-protecting groups) as defined herein for an alkyl.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like. In some embodiments, the hydroxyalkylgroup can be substituted with 1, 2, 3, or 4 substituent groups (e.g.,O-protecting groups) as defined herein for an alkyl.

The term “hydroxyalkynyl,” as used herein, represents an alkynyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group. In some embodiments, the hydroxyalkynylgroup can be substituted with 1, 2, 3, or 4 substituent groups (e.g.,O-protecting groups) as defined herein for an alkyl.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “O-protecting group,” as used herein, represents those groupsintended to protect an oxygen containing (e.g., phenol, hydroxyl, orcarbonyl) group against undesirable reactions during syntheticprocedures. Commonly used O-protecting groups are disclosed in Greene,“Protective Groups in Organic Synthesis,” 3^(rd) Edition (John Wiley &Sons, New York, 1999), which is incorporated herein by reference.Exemplary O-protecting groups include acyl, aryloyl, or carbamyl groups,such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl,tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl,phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and4-nitrobenzoyl; alkylcarbonyl groups, such as acyl, acetyl, propionyl,pivaloyl, and the like; optionally substituted arylcarbonyl groups, suchas benzoyl; silyl groups, such as trimethylsilyl (TMS),tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM),triisopropylsilyl (TIPS), and the like; ether-forming groups with thehydroxyl, such methyl, methoxymethyl, tetrahydropyranyl, benzyl,p-methoxybenzyl, trityl, and the like; alkoxycarbonyls, such asmethoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl,n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl,sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl,cyclohexyloxycarbonyl, methyloxycarbonyl, and the like;alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl,ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl,2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl,allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl,3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyls, such as2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, and the like; optionally substitutedarylalkoxycarbonyl groups, such as benzyloxycarbonyl,p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl,3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxy-carbonyl, fluorenylmethyloxycarbonyl, and the like; andoptionally substituted aryloxycarbonyl groups, such as phenoxycarbonyl,p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl,2,4-dinitrophenoxycarbonyl, p-methyl-phenoxycarbonyl,m-methylphenoxycarbonyl, o-bromophenoxycarbonyl,3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl,2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl,aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl; methoxymethyl;benzyloxymethyl; siloxymethyl; 2,2,2-trichloroethoxymethyl;tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl;1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether;p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl,and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl;triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl;t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; anddiphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl,9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl;vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl;and nitrobenzyl); carbonyl-protecting groups (e.g., acetal and ketalgroups, such as dimethyl acetal, 1,3-dioxolane, and the like; acylalgroups; and dithiane groups, such as 1,3-dithianes, 1,3-dithiolane, andthe like); carboxylic acid-protecting groups (e.g., ester groups, suchas methyl ester, benzyl ester, t-butyl ester, orthoesters, and the like;and oxazoline groups.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical. Perfluoroalkoxy groups areexemplified by trifluoromethoxy, pentafluoroethoxy, and the like.

The term “spirocyclyl,” as used herein, represents a C₂₋₇alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein, and the sulfo group can befurther substituted with one or more O-protecting groups (e.g., asdescribed herein).

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkaryl group. In someembodiments, the alkaryl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein.

The term “thioalkheterocyclyl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkheterocyclyl group. In someembodiments, the alkheterocyclyl group can be further substituted with1, 2, 3, or 4 substituent groups as described herein.

The term “thioalkoxy,” as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone—enol pairs, amide—imidicacid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the mRNA of the present invention may be single units or multimers orcomprise one or more components of a complex or higher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of apolynucleotide to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form multimers (e.g., through linkageof two or more polynucleotides) or conjugates, as well as to administera payload, as described herein. Examples of chemical groups that can beincorporated into the linker include, but are not limited to, alkyl,alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers,Other examples include, but are not limited to, cleavable moietieswithin the linker, such as, for example, a disulfide bond (—S—S—) or anazo bond (—N═N—), which can be cleaved using a reducing agent orphotolysis. Non-limiting examples of a selectively cleavable bondinclude an amido bond can be cleaved for example by the use oftris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/orphotolysis, as well as an ester bond can be cleaved for example byacidic or basic hydrolysis.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody. Patient: As used herein, “patient” refers to asubject who may seek or be in need of treatment, requires treatment, isreceiving treatment, will receive treatment, or a subject who is undercare by a trained professional for a particular disease or condition.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety.

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use ofprodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.

“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Significant or Significantly: As used herein, the terms “significant” or“significantly” are used synonymously with the term “substantially.”

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

EXAMPLES

The present disclosure is further described in the following examples,which do not limit the scope of the disclosure described in the claims.

Example 1 Modified mRNA In Vitro Transcription A. Materials and Methods

Modified mRNAs according to the invention are made using standardlaboratory methods and materials for in vitro transcription with theexception that the nucleotide mix contains modified nucleotides. Theopen reading frame (ORF) of the gene of interest is flanked by a 5′untranslated region (UTR) containing a strong Kozak translationalinitiation signal and an alpha-globin 3′ UTR terminating with anoligo(dT) sequence for templated addition of a polyA tail for mRNAs notincorporating adenosine analogs. Adenosine-containing mRNAs aresynthesized without an oligo (dT) sequence to allow forpost-transcription poly (A) polymerase poly-(A) tailing.

The modified mRNAs may be modified to reduce the cellular innate immuneresponse. The modifications to reduce the cellular response may includepseudouridine (ψ) and 5-methyl-cytidine (5meC, 5 mc or m⁵C). (See,Kariko K et al. Immunity 23:165-75 (2005), Kariko K et al. Mol Ther16:1833-40 (2008), Anderson B R et al. NAR (2010); herein incorporatedby reference).

The ORF may also include various upstream or downstream additions (suchas, but not limited to, β-globin, tags, etc.) may be ordered from anoptimization service such as, but limited to, DNA2.0 (Menlo Park,Calif.) and may contain multiple cloning sites which may have XbaIrecognition. Upon receipt of the construct, it may be reconstituted andtransformed into chemically competent E. coli.

For the present invention, NEB DH5-alpha Competent E. coli are used.Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10minutes.

Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture.Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at 42° C. for exactly 30 seconds. Do not mix.

Place on ice for 5 minutes. Do not mix.

Pipette 950 μl of room temperature SOC into the mixture.

Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 37° C.

Mix the cells thoroughly by flicking the tube and inverting.

Spread 50-100 μl of each dilution onto a selection plate and incubateovernight at 37° C. Alternatively, incubate at 30° C. for 24-36 hours or25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmid(an Example of which is shown in FIG. 3) is first linearized using arestriction enzyme such as XbaI. A typical restriction digest with XbaIwill comprise the following: Plasmid 1.0 μg; 10× Buffer 1.0 μl; XbaI 1.5μl; dH₂O up to 10 μl; incubated at 37° C. for 1 hr. If performing at labscale (<5 μg), the reaction is cleaned up using Invitrogen's PURELINK™PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions. Largerscale purifications may need to be done with a product that has a largerload capacity such as Invitrogen's standard PURELINK™ PCR Kit (Carlsbad,Calif.). Following the cleanup, the linearized vector is quantifiedusing the NanoDrop and analyzed to confirm linearization using agarosegel electrophoresis.

B. Agarose Gel Electrophoresis of Modified mRNA

Individual modified mRNAs (200-400 ng in a 20 μl volume) are loaded intoa well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad,Calif.) and run for 12-15 minutes according to the manufacturerprotocol.

C. Agarose Gel Electrophoresis of RT-PCR Products

Individual reverse transcribed-PCR products (200-400 ng) are loaded intoa well of a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad,Calif.) and run for 12-15 minutes according to the manufacturerprotocol.

D. Nanodrop Modified mRNA Quantification and UV Spectral Data

Modified mRNAs in TE buffer (1 μl) are used for Nanodrop UV absorbancereadings to quantitate the yield of each modified mRNA from an in vitrotranscription reaction (UV absorbance traces are not shown).

Example 2 Modified mRNA Transfection A. Reverse Transfection

For experiments performed in a 24-well collagen-coated tissue cultureplate, Keratinocytes are seeded at a cell density of 1×10⁵. Forexperiments performed in a 96-well collagen-coated tissue culture plate,Keratinocytes are seeded at a cell density of 0.5×10⁵. For each modifiedmRNA to be transfected, modified mRNA: RNAIMAX™ are prepared asdescribed and mixed with the cells in the multi-well plate within 6hours of cell seeding before cells had adhered to the tissue cultureplate.

B. Forward Transfection

In a 24-well collagen-coated tissue culture plate, Keratinocytes areseeded at a cell density of 0.7×10⁵. For experiments performed in a96-well collagen-coated tissue culture plate, Keratinocytes are seededat a cell density of 0.3×10⁵. Keratinocytes are then grown to aconfluency of >70% for over 24 hours. For each modified mRNA to betransfected, modified mRNA: RNAIMAX™ are prepared as described andtransfected onto the cells in the multi-well plate over 24 hours aftercell seeding and adherence to the tissue culture plate.

C. Modified mRNA Translation Screen G-CSF ELISA

Keratinocytes are grown in EpiLife medium with Supplement S7 fromInvitrogen at a confluence of >70%. Keratinocytes are reversetransfected with 300 ng of the indicated chemically modified mRNAcomplexed with RNAIMAX™ from Invitrogen. Alternatively, keratinocytesare forward transfected with 300 ng modified mRNA complexed withRNAIMAX™ from Invitrogen. The RNA: RNAIMAX™ complex is formed by firstincubating the RNA with Supplement-free EPILIFE® media in a 5×volumetric dilution for 10 minutes at room temperature.

In a second vial, RNAIMAX™ reagent is incubated with Supplement-freeEPILIFE® Media in a 10× volumetric dilution for 10 minutes at roomtemperature. The RNA vial is then mixed with the RNAIMAX™ vial andincubated for 20-30 at room temperature before being added to the cellsin a drop-wise fashion. Secreted huG-CSF concentration in the culturemedium is measured at 18 hours post-transfection for each of thechemically modified mRNAs in triplicate. Secretion of HumanGranulocyte-Colony Stimulating Factor (G-CSF) from transfected humankeratinocytes is quantified using an ELISA kit from Invitrogen or R&DSystems (Minneapolis, Minn.) following the manufacturers recommendedinstructions.

D. Modified mRNA Dose and Duration G-CSF ELISA

Keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70%. Keratinocytes are reversetransfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or1500 ng modified mRNA complexed with RNAIMAX™ from Invitrogen. Themodified mRNA: RNAIMAX™ complex is formed as described. Secreted huG-CSFconcentration in the culture medium is measured at 0, 6, 12, 24, and 48hours post-transfection for each concentration of each modified mRNA intriplicate. Secretion of Human Granulocyte-Colony Stimulating Factor(G-CSF) from transfected human keratinocytes is quantified using anELISA kit from Invitrogen or R&D Systems following the manufacturersrecommended instructions.

Example 3 Cellular Innate Immune Response to Modified Nucleic Acids:IFN-Beta ELISA and TNF-Alpha ELISA

An enzyme-linked immunosorbent assay (ELISA) for Human Tumor NecrosisFactor-α (TNF-α), Human Interferon-β (IFN-β) and HumanGranulocyte-Colony Stimulating Factor (G-CSF) secreted from invitro-transfected Human Keratinocyte cells is tested for the detectionof a cellular innate immune response.

Keratinocytes are grown in EPILIFE® medium with Human KeratinocyteGrowth Supplement in the absence of hydrocortisone from Invitrogen at aconfluence of >70%. Keratinocytes are reverse transfected with 0 ng,93.75 ng, 187.5 ng, 375 ng, 750 ng, 1500 ng or 3000 ng of the indicatedchemically modified mRNA complexed with RNAIMAX™ from Invitrogen asdescribed in triplicate. Secreted TNF-α in the culture medium ismeasured 24 hours post-transfection for each of the chemically modifiedmRNAs using an ELISA kit from Invitrogen according to the manufacturerprotocols.

Secreted IFN-β is measured 24 hours post-transfection for each of thechemically modified mRNAs using an ELISA kit from Invitrogen accordingto the manufacturer protocols. Secreted hu-G-CSF concentration ismeasured at 24 hours post-transfection for each of the chemicallymodified mRNAs. Secretion of Human Granulocyte-Colony Stimulating Factor(G-CSF) from transfected human keratinocytes is quantified using anELISA kit from Invitrogen or R&D Systems (Minneapolis, Minn.) followingthe manufacturers recommended instructions. These data indicate whichmodified mRNA are capable eliciting a reduced cellular innate immuneresponse in comparison to natural and other chemically modifiedpolynucleotides or reference compounds by measuring exemplary type 1cytokines TNF-alpha and IFN-beta.

Example 4 Human Granulocyte-Colony Stimulating Factor-ModifiedmRNA-Induced Cell Proliferation Assay

Human keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70% in a 24-well collagen-coatedTRANSWELL® (Corning, Lowell, Mass.) co-culture tissue culture plate.Keratinocytes are reverse transfected with 750 ng of the indicatedchemically modified mRNA complexed with RNAIMAX™ from Invitrogen asdescribed in triplicate. The modified mRNA: RNAIMAX™ complex is formedas described. Keratinocyte media is exchanged 6-8 hourspost-transfection. 42-hours post-transfection, the 24-well TRANSWELL®plate insert with a 0.4 μm-pore semi-permeable polyester membrane isplaced into the hu-G-CSF modified mRNA-transfected keratinocytecontaining culture plate.

Human myeloblast cells, Kasumi-1 cells or KG-1 (0.2×10⁵ cells), areseeded into the insert well and cell proliferation is quantified 42hours post-co-culture initiation using the CyQuant Direct CellProliferation Assay (Invitrogen) in a 100-120 μl volume in a 96-wellplate. modified mRNA-encoding hu-G-CSF-induced myeloblast cellproliferation is expressed as a percent cell proliferation normalized tountransfected keratinocyte/myeloblast co-culture control wells. Secretedhu-G-CSF concentration in both the keratinocyte and myeloblast insertco-culture wells is measured at 42 hours post-co-culture initiation foreach modified mRNA in duplicate. Secretion of Human Granulocyte-ColonyStimulating Factor (G-CSF) is quantified using an ELISA kit fromInvitrogen following the manufacturers recommended instructions.

Transfected hu-G-CSF modified mRNA in human keratinocyte feeder cellsand untransfected human myeloblast cells are detected by RT-PCR. TotalRNA from sample cells is extracted and lysed using RNAEASY® kit (Qiagen,Valencia, Calif.) according to the manufacturer instructions. Extractedtotal RNA is submitted to RT-PCR for specific amplification of modifiedmRNA-G-CSF using PROTOSCRIPT® M-MuLV Taq RT-PCR kit (New EnglandBioLabs, Ipswich, Mass.) according to the manufacturer instructions withhu-G-CSF-specific primers. RT-PCR products are visualized by 1.2%agarose gel electrophoresis.

Example 5 Cytotoxicity and Apoptosis

This experiment demonstrates cellular viability, cytotoxity andapoptosis for distinct modified mRNA-in vitro transfected HumanKeratinocyte cells. Keratinocytes are grown in EPILIFE® medium withHuman Keratinocyte Growth Supplement in the absence of hydrocortisonefrom Invitrogen at a confluence of >70%. Keratinocytes are reversetransfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng,1500 ng, 3000 ng, or 6000 ng of modified mRNA complexed with RNAIMAX™from Invitrogen. The modified mRNA: RNAIMAX™ complex is formed. SecretedhuG-CSF concentration in the culture medium is measured at 0, 6, 12, 24,and 48 hours post-transfection for each concentration of each modifiedmRNA in triplicate. Secretion of Human Granulocyte-Colony StimulatingFactor (G-CSF) from transfected human keratinocytes is quantified usingan ELISA kit from Invitrogen or R&D Systems following the manufacturersrecommended instructions. Cellular viability, cytotoxicity and apoptosisis measured at 0, 12, 48, 96, and 192 hours post-transfection using theAPOTOX-GLO™ kit from Promega (Madison, Wis.) according to manufacturerinstructions.

Example 6 Co-Culture Environment

The modified mRNA comprised of chemically-distinct modified nucleotidesencoding human Granulocyte-Colony Stimulating Factor (G-CSF) maystimulate the cellular proliferation of a transfection incompetent cellin co-culture environment. The co-culture includes a highlytransfectable cell type such as a human keratinocyte and a transfectionincompetent cell type such as a white blood cell (WBC). The modifiedmRNA encoding G-CSF may be transfected into the highly transfectablecell allowing for the production and secretion of G-CSF protein into theextracellular environment where G-CSF acts in a paracrine-like manner tostimulate the white blood cell expressing the G-CSF receptor toproliferate. The expanded WBC population may be used to treatimmune-compromised patients or partially reconstitute the WBC populationof an immunosuppressed patient and thus reduce the risk of opportunisticinfections.

In another example, a highly transfectable cell such as a fibroblast aretransfected with certain growth factors to support and simulate thegrowth, maintenance, or differentiation of poorly transfectableembryonic stem cells or induced pluripotent stem cells.

Example 7 5′-Guanosine Capping on Modified Nucleic Acids (modifiedmRNAs) A. Materials and Methods

The cloning, gene synthesis and vector sequencing was performed byDNA2.0 Inc. (Menlo Park, Calif.). The ORF was restriction digested usingXbaI and used for cDNA synthesis using tailed-or tail-less-PCR. Thetailed-PCR cDNA product was used as the template for the modified mRNAsynthesis reaction using 25 mM each modified nucleotide mix (allmodified nucleotides were custom synthesized or purchased from TriLinkBiotech, San Diego, Calif. except pyrrolo-C triphosphate purchased fromGlen Research, Sterling Va.; unmodified nucleotides were purchased fromEpicenter Biotechnologies, Madison, Wis.) and CellScript MEGASCRIPT™(Epicenter Biotechnologies, Madison, Wis.) complete mRNA synthesis kit.The in vitro transcription reaction was run for 4 hours at 37° C.Modified mRNAs incorporating adenosine analogs were poly (A) tailedusing yeast Poly (A) Polymerase (Affymetrix, Santa Clara, Calif.). PCRreaction used HiFi PCR 2× MASTER MIX™ (Kapa Biosystems, Woburn, Mass.).Modified mRNAs were post-transcriptionally capped using recombinantVaccinia Virus Capping Enzyme (New England BioLabs, Ipswich, Mass.) anda recombinant 2′-o-methyltransferase (Epicenter Biotechnologies,Madison, Wis.) to generate the 5′-guanosine Cap 1 structure. Cap 2structure and Cap 2 structures may be generated using additional2′-o-methyltransferases. The In vitro transcribed mRNA product was runon an agarose gel and visualized. Modified mRNA was purified withAmbion/Applied Biosystems (Austin, Tex.) MEGAClear RNA™ purificationkit. PCR used PURELINK™ PCR purification kit (Invitrogen, Carlsbad,Calif.). The product was quantified on NANODROP™ UV Absorbance(ThermoFisher, Waltham, Mass.). Quality, UV absorbance quality andvisualization of the product was performed on an 1.2% agarose gel. Theproduct was resuspended in TE buffer.

B. 5′ Capping Modified Nucleic Acid (mRNA) Structure

5′-capping of modified mRNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m⁷G(5′)ppp(5′)G (the ARCA cap);G(5′)ppp(5′)A; G(5′)ppp(5′)G; m⁷G(5′)ppp(5′)A; m⁷G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified mRNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m⁷G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-o-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-o-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of 12-18 hours or more than 18 hours, e.g., 24, 36, 48, 60, 72or greater than 72 hours.

Example 8 Synthesis of N4-Methyl Cytidine (Compound 1) and N4-Methyl CTP(NTP of Said Compound)

Uridine was silylated to provide a trisilylated compound, which waspurified by column, activated with re-distilled POCl₃/triazole underanhydrous condition, and then followed by nucleophilic substitution with40% methylamine aqueous solution.N4-Methyl-2′,3′,5′-tri-O-TBDMS-cytidine was thus obtained afterchromatographic purification. The resultant product was deprotected withTBAF and then purified with an ethanol-ethyl acetate (3:1) solventsystem to obtain compound 1. The final product was characterized by NMR(in DMSO); MS: 258 (M+H)⁺, 280 (M+Na)⁺, and 296 (M+K)⁺; and HPLC:purity, 99.35% (FIGS. 1A-1D). HPLC, purity 98% (FIG. 2).

Example 9 Synthesis of 2′-OMe-N,N-di-Me-Cytidine (Compound 2) and2′-OMe-N,N-Di-Me-CTP (NTP of Said Compound)

2′-O-Methyluridine was silylated to give the di-silylated compound.Purified 2′-O-methyl-3′,5′-di-O-TBDMS uridine was activated withre-distilled POCl₃ and imidazole under anhydrous condition, followed bythe nucleophilic substitution with dimethylamine hydrochloride undertriethylamine environment to trap HCl. Intermediate compoundN4,N4,2′-tri-O-methyl-3′,5′-bis-O-TBDMS uridine was purified by flashchromatography and obtained as a white foam. The resultant compound wasde-protected with TBAF and then purified to provide ˜400 mg finalproduct compound 2 as white foam. ES MS: m/z 308 (M+Na)⁺, 386 (M+H)⁺;HPLC: purity, 99.49% (FIGS. 3A-3C).

To synthesize the corresponding NTP, 70 mg of nucleoside compound 2provided 23 mg of 2′-OMe-N,N-di-Me-CTP after purification viaion-exchange and reverse phase columns. HPLC: purity, 95% (FIG. 4).

Example 10 Synthesis of 5-Methoxycarbonylmethoxy Uridine (Compound 3)and 5-Methoxycarbonylmethoxy-UTP (NTP of Said Compound)

Uridine 3-a in water was treated with excess amount of bromine and thenflushed with air to remove bromine. The reaction mixture was treatedwith pyridine at a controlled speed and temperature. During thereaction, unstable bromo-intermediate 3-b gradually converted todi-hydroxyl intermediate 3-c, which presumably dehydrated to the stable5-hydroxyuridine 3-d. Then, the 5-hydroxyuridine was protected with a2′,3′-isopropylidene group to provide compound 3-g. Reaction withcompound 3-f provided compound 3.

60-70 mg of the nucleoside provided >21 mg of the desired triphosphateafter two HPLC column purification and two lyophilization steps. HPLC:purity, 98% (FIG. 5).

Example 11 Synthesis of 3-Methyl Pseudouridine (Compound 4) and 3-MethylPseudo-UTP (NTP of Said Compound)

Pseudouridine 4-a was reacted with Ac₂O to provide acetyl-protectedpseudouridine 4-b. Then, N1 was selectively protected with POM toprovide compound 4-c. Methylation of N3, followed by deprotected,provided compound 4 (˜400 mg). Molecular formula: C10H14N2O6, molecularweight: 258.23 g/mol; appearance: white solid; storage conditions: storeat 25° C.; HPLC: purity, 98.51%; ¹H NMR (DMSO-d₆): δ 11.17 (d, 1H, J=3.0Hz), 7.56 (d, 1H, J=3.6 Hz), 4.91 (d, 1H, J=3.6 Hz), 4.79 (t, 1H, J=4.2Hz), 4.70 (d, 1H, J=4.2 Hz), 4.49 (d, 1H, J=3.0 Hz), 3.82-3.88 (m, 2H),3.66-3.67 (m, 1H), 3.57-3.61 (m, 1H), 3.40-3.47 (m, 1H), 3.09 (s, 3H);MS: 281 (M+Na)) (FIGS. 6A and 6B).

Alternative routes could be applied to obtain compound 4. For example,pseudouridine could be reacted with an O-protecting group (e.g., asdescribed herein, such as TMS) and reacted with an N-protecting group(e.g., as described herein, such as acetyl at N1). Then, N3 of thenucleobase could be reacted with an alkylating agent (e.g.,dimethylamine/dimethoxymethyl) to provide compound 4 having N- andO-protecting groups. Finally, the resultant compound would bedeprotected (e.g., under basic conditions, such as NH₃/MeOH) to providecompound 4.

Example 12 Synthesis of N—Ac, 5—Ac—OCH₂-Cytidine (Compound 5)

Uridine 5-a was protected to obtain isopropylidene compound 5-b, whichwas reacted with (CHCO)_(n). Acetic acid with catalyst amount of TFA wasemployed to obtain the desired selectively acylated compound 5-f (30%yield). Further tritylation of the 5′-OH group resulted in the desiredorthogonally protected compound 5-g.

Compound 5-g was treated with POCl₃ and triazole to provide compound 5-htogether with de-acylated compound 5-i. Acetylation of these twocompounds provided di-acylated, fully protected compound 5-j.Deprotection of compound 5-j with acetic acid under heating conditionresulted in three products, one of which was compound 5.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Alternative routes could be applied to obtain compound 5, such as bybeginning with cytidine as the starting material. In such methods, the5-position could be reacted with a halogen or a halogenation agent(e.g., any described herein, such as I₂/meta-chloroperoxybenzoic acid),which can be displaced with an alkylating agent. Further, such methodscould include the use of one or more N- or O-protecting groups (e.g.,any described herein, such as silylation or acetylation) to protect theamino group of cytidine and/or hydroxyl groups of the sugar moiety.

Example 13 Synthesis of 5-TBDMS-OCH₂-cytidine (Compound 6)

A 5-hydroxyuracil compound ′-b was glycosylated to obtain compound 6′-d(28% yield), which was silylated to provide compound 6′-e. Activation ofthe protected uridine provided the desired compound 6 after furtheramination and deprotection (800 mg of the final compound). Molecularformula: C16H29N3O6Si; molecular weight: 387.50 g/mol; appearance: whitesolid; storage conditions: store at 25° C.; HPLC: purity, 97.57%; ¹H NMR(CDCl₃): d 7.81 (s, 1H), 7.40 (bs, 1H), 6.49 (bs, 1H), 5.79 (d, 1H,J=2.4 Hz), 5.3-5.32 (m, 1H), 5.00-5.07 (m, 2H), 4.30-4.45 (m, 2H),3.90-3.94 (m, 2H), 3.80-3.83 (m, 1H), 3.50-3.70 (m, 2H), 0.87 (s, 9H),0.05 (S, 6H); MS: 388 (M+H)⁺, 410 (M+Na)⁺) (FIGS. 7A-7C).

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 14 Synthesis of 5-trifluoromethyl cytidine (Compound 7)

Compound 7-A was glycosylated to provide compound 7-B, which was treatedwith 2,4,6-triisopropylbenzene sulfonyl chloride (TPSCl) to activate thecarbonyl group and to promote reductive amination. Deprotection providedcompound 7. Alternative activating agents could be used instead ofTPSCl, such as 2,4,6-trimethylbenzene sulfonyl chloride.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 15 Synthesis of 5-trifluoromethyl uridine (Compound 8)

5-Trifluoromethyluracil 8-A was glycosylated with tetra-O-acetyl ribose,and the desired triprotected 5-trifluoromethyluridine 8-B was obtainedin good yield. Further deprotection gave desired compound 8, which wascharacterized with NMR, MS and HPLC results. MS: 313 (M+H)⁺, 335(M+Na)⁺; HPLC: purity, 98.87%, ((FIGS. 8A-8C).

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 16 Synthesis of 5-(methoxycarbonyl)methyl uridine (Compound 9)

Uridine 9-a was protected to provide compound 9-b (98% yield). Thiscompound was brominated with excess bromine in the presence of aceticanhydride and acetic acid. The 5-bromo analog 9-c was obtained (60%yield) and further benzoylated to provide desired compound 9-d (64%yield). 5-Bromo compound 9-d was condensed with dimethyl malonate underbasic condition to give the arylated malonate and the fully protecteddiester 9-e (50% yield). After de-carboxylation and deprotection,compound 9 was obtained verified by NMR (FIG. 9).

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 17 Synthesis of 5-(methoxycarbonyl)methyl-2′-O-methyl uridine(2-OMe-MCM5U) (Compound 10)

Similar strategy to the synthesis of compound 9 above,2′-O-methyluridine 10-a was acylated and brominated to obtain compound10-c. Further benzoylation provided 5-bromo analog 10-d, which wascondensed with dimethyl malonate provide the desired product 10-e (45%yield). Decarboxylation and deprotection provided compound 10.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 18 Synthesis of 5-trifluoroacetyl-aminomethyl-2-thiouridine(Compound 11)

Glycosylation of 2-thiouracil 11-a provided compound 11-c, which can bedeprotected with any useful deprotection reagent. In particular, LiOHprovided desired product 11-d (80-90% yield). Isopropylidene protectionprovided compound 11-e (90% yield). Further 5-hydroxylmethylationprovided compound 11-f. Chlorination, azidation, and further reductionprovided methylamine compound 11-i, which was acetylated to providedcompound 11.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 19 Synthesis of 5-methylaminomethyl-2-uridine (Compound 12)

Compound 12 can be obtained by any useful method (e.g., see schemes (i)and (ii) above). For example, protected uracil can be glycosylated andsubsequently aminated to provide compound 12. Additional protecting,deprotecting, and activating steps can be conducted as needed. To obtainthe corresponding NTP, a triphosphate reaction can be conducted (e.g.,any described herein). Optionally, the NTP can be purified (e.g., usinga Sephadex DEAE-A25 column), lyophilized, or evaporated (e.g., fromEtOH).

Example 20 Synthesis of 5-TFA-methylaminomethyl-2-uridine (Compound 13)

Uridine 13-a was protected with isopropylidene to provide compound 13-band then 5-hydroxymethylated to provide compound 13-c. Chlorination andsubsequent amination provided compound 13-e, which can be protected toprovided 13-f. Subsequent deprotection provided compound 13.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 21 Synthesis of 5-carboxymethylaminomethyl uridine (Compound 14)

Uridine 14-a was protected with isopropylidene to provide compound 14-band then 5-aminoalkylated with the Mannich reaction to provide compound14-c. Methylation provided quaternary amine 14-d. Subsequent aminationand deprotection steps can be used to provide compound 14. To obtain thecorresponding NTP, a triphosphate reaction can be conducted (e.g., anydescribed herein). Optionally, the NTP can be purified (e.g., using aSephadex DEAE-A25 column), lyophilized, or evaporated (e.g., from EtOH).

Example 22 Alternative synthesis of 5-methylaminomethyl-2-uridine(Compound 12) and 5-carboxymethylaminomethyl-2-uridine (Compound 14)

In addition to those strategies provided above for compounds 12 and 14,the following strategy can also be implemented. 5-Methyluridine A can besilylated to provide compound B. After radical monobromination, theresultant intermediate bromide C can be used for the preparation ofcompound 12 and compound 14 analogs. Subsequent alkylamination ofbromide compound C could provide compounds D and E, which can bedeprotected to provide compounds 14 and 12, respectively. To obtain thecorresponding NTP, a triphosphate reaction can be conducted (e.g., anydescribed herein). Optionally, the NTP can be purified (e.g., using aSephadex DEAE-A25 column), lyophilized, or evaporated (e.g., from EtOH).

Example 23 Synthesis of Dimethyl-Pseudouridine (Compound 15) andDimethyl-Pseudo-UTP (NTP of Said Compound)

Nucleosides can be phosphorylated by any useful method. For example, asshown above, nucleosides can be reacted with phosphorus oxychloride andsubsequently treated with a monophosphate intermediate withbis(tributylammonium)pyrophosphate (TBAPP) to give the triphosphate.

Example 24 Synthesis of 2′-C-methyl adenosine (compound 16) and2′-C-methyl ATP (NTP of said compound)

About 5 g of compound 16-2 was prepared from 5 g of compound 16-1 via aDess-Martin periodane reaction. Compound 16-2 was reacted withMeMgI/TiCl4/−78° C. to provide compound 16-3, and crude compound 16-3 (6g) was directly reacted with benzylchloride to prepare compound 16-4.Reaction with the nucleobase and deprotection provided compound 16 (0.56g).

Example 25 Synthesis of 2′-C-methyl-cytidine isomers (compound 17 andcompound 18) and 2′-C-methyl UTP (NTP of said compounds)

About 17.4 g of compound 17-3 was prepared from 20 g of compound 17-1.Then, 2′-oxidation and alkylation with MeMgI provided 300 mg of compound17-5a and 80 mg of compound 17-5b. About 9 g of compound 17-5a (about90% pure) and 2.1 g of compound 17-5b (pure) were prepared from 17.4 gof compound 17-3 in 2 batches. N- and O-deprotection provided compounds17 and 18.

Example 26 Synthesis of 2′-C-methyl guanosine (compound 19) and2′-C-methyl GTP (NTP of said compound)

2′-Oxidation of protected ribose 19-1 and subsequent alkylation withMeMgCl provided compound 19-3. The resultant compound was furtherprotected to provided compound 19-4, and 1.56 g of compound 19-5a wasprepared from 3.1 g of compound 19-4. Subsequent oidation anddeprotection provided compound 19 (about 90% pure, 50 mg).

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 27 Synthesis of 2′-C-methyl uridine (compound 20) and2′-C-methyl UTP (NTP of said Compound)

2′-Oxidation of protected ribose 20-1 and subsequent alkylation withMeMgCl provided compound 20-3. The resultant compound was furtherprotected to provide compound 20-4. Reaction with uracil anddeprotection provided pure compound 20 (50 mg).

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 28 Synthesis of (S)-2′-C-methyl adenosine (compound 21) and(S)-2′-C-methyl ATP (NTP of said compound)

Compound 21-1 (5 g) was protected to form compound 21-2a, and chromiumoxidation provided compound 21-3a. Alkylation via route [i](5 eq. MeMgIin ether at −50° C.) provided compound 21-4. Optionally, yield could beimproved via route [ii] by protecting the amino group to providecompound 21-3b and then alkylating at the 2′-C position to providecompound 21-4-a. Compound 21-3a was alkylated to provide crude compound21-4 (3 g, 20% of compound 3a in this crude product), where the productcan be optionally purified. Deprotection of compound 21-4 affordedcompound 21 (50% yield).

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 29 Synthesis of (S)-2′-C-methyl guanosine (compound 22) and(S)-2′-methyl GTP (NTP of said Compound)

About 30 g of compound 22-1 was silylated to provide compound 22-2 inthree steps. Further protection provided compound 22-3, and Dess-Martinperiodane oxidation provided compound 22-4 (1.6 g) in two batches. 2′-Calkylation (5 eq. MeMgI in ether, −50° C. to RT) provided compound 22-5,and further deprotection steps provided compound 22.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 30 Synthesis of (S)-2′-C-methyl uridine (compound 23) and of(S)-2′-C-methyl UTP (NTP of said compound)

Uridine 23-1 (2.0 g) was protected with TIPDSCl₂(1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane) to provide compound23-2. Oxidation provided compound 23-3, and 2′-C alkylation providedcompound 23-4, which can be optionally purified with Prep-HPLC prior tothe next step. Then, deprotection provided desired compound 23.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 31 Synthesis of 4′-C-methyl adenosine (compound 24) and4′-C-methyl ATP (NTP of said Compound)

1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose 24-1 was converted viasequential oxidation, reduction, and protection steps to providecompound 24-4. The first oxidation step to provide compound 24-2 can beimplemented with any useful reagents, such as 0.75 eq. pyridiniumdichromate (PDC) with 1 eq. Ac₂O or 1.2 eq. of Dess-Martin periodane.Subsequent deprotection, formylation, and reduction provided compound24-7, which was followed with protection and deoxygenation steps toprovide compound 24-10. About 0.4 g of compound 24-14 was prepared from1 g of compound 24-10 via sequential protection and deprotection steps.Addition of N6-benzoyladenine and subsequent deprotection providedcompound 24.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 32 Synthesis of 4′-C-methyl cytidine (compound 25) and4′-C-methyl CTP (NTP of said compound)

Similar to the strategy provided above for compound 24, compound 25-14was produced with compound 25-1. Addition of cytidine and subsequentdeprotection provided compound 25.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 33 Synthesis of 4′-C-methyl guanosine (compound 26) and4′-C-methyl GTP (NTP of said compound)

Similar to the strategy provided above for compound 24, compound 26-14was produced with compound 26-1. Addition of 2-amino-6-chloropurine,subsequent oxidation, and then deprotection provided compound 26.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 34 Synthesis of 4′-C-methyl uridine (compound 27) and4′-C-methyl UTP (NTP of said compound)

Similar to the strategy provided above for compound 24, compound 27-14was produced with compound 27-1. Addition of uracil and subsequentdeprotection provided compound 27.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 35 Synthesis of 2′-O,4′-C-methylene adenosine (compound 28) and2′-O,4′-C-methylene ATP (NTP of said compound)

Similar to the strategy provided above for compound 24, compound 28-7was produced with compound 28-1. Subsequent mesylation, deprotection,and acetylation provided compound 28-10, which was followed by additionof N6-benzoyladenine and subsequent internal cyclization. Variousprotection and deprotection steps provided compound 28.

Example 36 Synthesis of 5-methyl-2′-O,4′-C-methylene cytidine (compound29) and 5-methyl-2′-O,4′-C-methylene CTP (NTP of said compound)

Aldofuranose compound 29-1 was reacted via various protection steps, andthen 5-methyluracil was added to provide compound 29-5. Subsequentinternal cyclization, deprotection, protection, and amination stepsprovided compound 29.

Example 37 Synthesis of 2′-O,4′-C-methylene guanosine (compound 30) and2′-O,4′-C-methylene GTP (NTP of said compound)

Similar to the strategy provided above for compound 29, aldofuranosecompound 30-1 was reacted via various protection steps, and then2-amino-6-chloropurine was added to provide compound 30-5. Subsequentinternal cyclization, amination, and deprotection steps providedcompound 30.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 38 Synthesis of 2′-O,4′-C-methylene uridine (compound 31) and2′-O,4′-C-methylene UTP (NTP of said compound)

Similar to the strategy provided above for compound 24, compound 31-7was produced with compound 31-1. Subsequent mesylation, deprotection,and acetylation provided compound 30-10. Addition of uracil andsubsequent internal cyclization provided compound 31-12, and variousprotection and deprotection steps provided compound 31. A subsequenttriphosphate reaction (e.g., as described herein) provided the NTP ofcompound 31, which can be optionally purified (e.g., with HPLC).

Example 39 Synthesis of 2′-chloro adenosine (compound 32) and 2′-chloroATP (NTP of said compound)

Arabinoadenosine 32-1 was protected via steps 1 and 2 and thenchlorinated to provide compound 32-4. Subsequent deprotection providedcompound 32, and the triphosphate reaction provided the NTP of compound32.

Example 40 Synthesis of 2′-iodo adenosine (compound 33) and 2′-iodo ATP(NTP of said compound)

Arabinoadenosine 33-1 was protected via steps 1 and 2 and then iodinatedto provide compound 33-4. Subsequent deprotection provided compound 33,and the triphosphate reaction in DMF provided the NTP of compound 33.

Example 41 Synthesis of 2′-bromo cytidine (compound 34) and 2′-bromo CTP(NTP of said compound)

Arabinocytidine 34-1 was protected under various conditions and thenbrominated to provide compound 34-4. Optionally, the reaction canprovide compound 34-4 via compound 34-3a under any useful protectionreactions, such as (i) 1.5 eq. Et₃N, 1 eq. DMAP, 1.2 eq. TfCl, in DCM(10 mL); (ii) 3 eq. DMAP, 1.2 eq. TfCl in DCM (15 mL); or (iii) 15 eq.DMAP, 1.5 eq. Tf₂O, in DCM (15 mL) at −10° C. to 0° C. for 2 hour. Inparticular, 55 mg of compound 34-3a was obtained from reaction condition(iii). Subsequent deprotection provided compound 34, and thetriphosphate reaction in DMF provided the NTP of compound 34. Crudeproduct 34 could be optionally purified prior to phosphorylation.

Example 42 Synthesis of 2′-chloro guanosine (compound 35) and 2′-chloroGTP (NTP of said compound)

Guanosine 35-1 was protected under various conditions and thenacetylated to provide compound 35-4. The reaction from compound 35-2 tocompound 35-3 was conducted with 2 eq. DMAP, 2 eq. Et₃N, 3 eq. Tf₂O in1,2-dichloroethane (10 mL) at 40° C. for 4 hours. About 55 mg ofcompound 35-3 was obtained after the purification.

Desired compound 35 can be obtained by any useful method. For example,as shown above, compound 35-4 can be treated with subsequent protection,chlorination, and deprotection steps to provide compound 35. To obtainthe corresponding NTP, a triphosphate reaction can be conducted (e.g.,any described herein). Optionally, the NTP can be purified (e.g., usinga Sephadex DEAE-A25 column), lyophilized, or evaporated (e.g., fromEtOH).

Example 43 Synthesis of 2′-iodo uridine (compound 36) and 2′-iodo UTP(NTP of said compound)

O²,2′-Cyclouridine 36-1 was protected to provide compound 36-2.Subsequent iodination, optionally mediated with selenium, providedcompound 36. A triphosphate reaction was conducted to provide the NTP ofcompound 36. Optionally, the NTP can be purified (e.g., using a SephadexDEAE-A25 column), lyophilized, or evaporated (e.g., from EtOH).

Example 44 Synthesis of 2′-O,4′-C-methylene adenosine (compound 37) and2′-O,4′-C-methylene ATP (NTP of said compound)

Similar to the strategy provided above for compound 24, compound 37-7was produced with compound 37-1. Subsequent mesylation, deprotection,and acetylation provided compound 37-10. Addition of uracil andsubsequent internal cyclization provided compound 37-12. Variousprotection and deprotection steps provided compound 37.

To obtain the corresponding NTP, a triphosphate reaction can beconducted (e.g., any described herein). Optionally, the NTP can bepurified (e.g., using a Sephadex DEAE-A25 column), lyophilized, orevaporated (e.g., from EtOH).

Example 45 Synthesis of cyclopentene diol cytidine (compound 38) andcyclopentene diol CTP (NTP of said compound)

D-ribose was protected and then allylated to provide compound 38-4,which was subsequently cyclized and reduced to provide compound 38-7.Olefin metathesis and subsequent oxidation provided compound 38-9, andfurther reduction reactions and addition of N-benzoyluracil providedcompound 38-14. Additional deprotection and protection reactionsprovided compound 38, and triphosphate reaction (e.g., with any usefulreaction condition, such as those described herein or in U.S. Pat. No.7,893,227, incorporated herein by reference) provided the NTP ofcompound 38.

Example 46 Synthesis of 2′-methyl uridine (compound 39) and 2′-methylUTP (NTP of said compound)

Uridine 39-1 was protected and then oxidized with 2 eq. of Dess-Martinperiodane to provide compound 39-3. Subsequent Wittig reaction,hydrogenation, and deprotection steps provided compound 39.

Example 47 Synthesis of 2′-methyl cytidine (compound 40) and 2′-methylCTP (NTP of said compound)

Cytidine 40-1 was protected and then oxidized to provide compound 40-3.Subsequent Wittig reaction, hydrogenation, and deprotection stepsprovided compound 40.

Example 48 Synthesis of N-acetyl cytidine (compound 41) and N-acetyl CTP(NTP of said compound)

A solution of N-acetyl-cytidine (compound 41) (103.0 mg, 0.36 mmol) wasadded to proton sponge (115.72 mg, 0.54 mmol, 1.50 equiv) in 1.0 mLtrimethylphosphate (TMP) and 1.0 mL of anhydrous tetrahydrofuran (THF).The solution was stirred for 10 minutes at 0° C. Phosphorous oxychloride(POCl₃) (67.2 ul, 0.72 mmol, 2.0 eqiv.) was added dropwise to thesolution before being kept stirring for 2 hours under N₂ atmosphere.After 2 hours the solution was reacted with a mixture ofbistributylammonium pyrophosphate (TBAPP or (n-Bu₃NH)₂H₂P₂O₇) (1.28 g,2.34 mmol, 6.5 eqiv.) and tributylamine (350.0 ul, 1.45 mmol, 4.0equiv.) in 2.5 ml of dimethylformamide. After approximately 15 minutes,the reaction was quenched with 24.0 ml of 0.2M triethylammoniumbicarbonate (TEAB) and the clear solution was stirred at roomtemperature for an hour. The reaction mixture was lyophilized overnightand the crude reaction mixture was purified by HPLC (Shimadzu, KyotoJapan, Phenomenex C18 preparative column, 250×21.20 mm, 10.0 micron;gradient: 100% A for 3.0 min, then 1% B/min, A=100 mM TEAB buffer,B=ACN; flow rate: 10.0 mL/min; retention time: 16.81-17.80 min).Fractions containing the desired compound were pooled and lyophilized toproduce the NTP of compound 41. The triphosphorylation reactions werecarried out in a two-neck flask flame-dried under N₂ atmosphere.Nucleosides and the protein sponge were dried over P₂O₅ under vacuumovernight prior to use. The formation of monophosphates was monitored byLCMS.

Example 49 Synthesis of 5-methoxy uridine (compound 42) and 5-methoxyUTP (NTP of said compound)

A solution of 5-methoxy uridine (compound 42) (69.0 mg, 0.25 mmol, plusheat to make it soluble) was added to proton sponge (80.36 mg, 0.375mmol, 1.50 equiv.) in 0.7 mL trimethylphosphate (TMP) and was stirredfor 10 minutes at 0° C. Phosphorous oxychloride (POCl₃) (46.7 ul, 0.50mmol, 2.0 equiv.) was added dropwise to the solution before being keptstirring for 2 hours under N₂ atmosphere. After 2 hours the solution wasreacted with a mixture of bistributylammonium pyrophosphate (TBAPP or(n-Bu₃NH)₂H₂P₂O₇) (894.60 mg, 1.63 mmol, 6.50 equiv.) and tributylamine(243.0 ul, 1.00 mmol, 4.0 equiv.) in 2.0 ml of dimethylformamide. Afterapproximately 15 minutes, the reaction was quenched with 17.0 ml of 0.2Mtriethylammonium bicarbonate (TEAB) and the clear solution was stirredat room temperature for an hour. The reaction mixture was lyophilizedovernight and the crude reaction mixture was purified by HPLC (Shimadzu,Kyoto Japan, Phenomenex C18 preparative column, 250×21.20 mm, 10.0micron; gradient: 100% A for 3.0 min, then 1% B/min, A=100 mM TEABbuffer, B=ACN; flow rate: 10.0 mL/min; retention time: 16.57-17.51 min).Fractions containing the desired compound were pooled and lyophilized toproduce the NTP of compound 42. The triphosphorylation reactions werecarried out in a two-neck flask flame-dried under N₂ atmosphere.Nucleosides and the protein sponge were dried over P₂O₅ under vacuumovernight prior to use. The formation of monophosphates was monitored byLCMS.

Example 50 Synthesis of 5-formyl cytidine (compound 43) and 5-formyl CTP(NTP of said compound)

A solution of 5-formyl cytidine (compound 43)) (48.4 mg, 0.18 mmol, plusheat to make it soluble) was added to proton sponge (57.86 mg, 0.27mmol, 1.50 equiv.) in 0.7 mL trimethylphosphate (TMP) and was stirredfor 10 minutes at 0° C. Phosphorous oxychloride (POCl₃) (33.6 ul, 0.36mmol, 2.0 equiv.) was added dropwise to the solution before being keptstirring for 2 hours under N₂ atmosphere. After 2 hours the solution wasreacted with a mixture of bistributylammonium pyrophosphate (TBAPP or(n-Bu₃NH)₂H₂P₂O₇) (642.0 mg, 1.17 mmol, 6.50 equiv.) and tributylamine(175.0 ul, 0.72 mmol, 4.0 equiv.) in 1.7 ml of dimethylformamide. Afterapproximately 15 minutes, the reaction was quenched with 12.0 ml of 0.2Mtriethylammonium bicarbonate (TEAB) and the clear solution was stirredat room temperature for an hour. The reaction mixture was lyophilizedovernight and the crude reaction mixture was purified by HPLC (Shimadzu,Kyoto Japan, Phenomenex C18 preparative column, 250×21.20 mm, 10.0micron; gradient: 100% A for 3.0 min, then 1% B/min, A=100 mM TEABbuffer, B=ACN; flow rate: 10.0 mL/min; retention time: 17.04-17.87 min).Fractions containing the desired compound were pooled and lyophilized toprovide the NTP of compound 43. The triphosphorylation reactions werecarried out in a two-neck flask flame-dried under N₂ atmosphere.Nucleosides and the protein sponge were dried over P₂O₅ under vacuumovernight prior to use. The formation of monophosphates was monitored byLCMS.

Example 51 Synthesis of 3-methyl uridine (compound 44) and 3-methyl UTP(NTP of said compound)

A solution of 3-methyl uridine (compound 44) (45.80 mg, 0.18 mmol) wasadded to proton sponge (57.86 mg, 0.27 mmol, 1.50 equiv.) in 0.5 mLtrimethylphosphate (TMP) and was stirred for 10 minutes at 0° C.Phosphorous oxychloride (POCl₃) (33.6 ul, 0.36 mmol, 2.0 equiv.) wasadded dropwise to the solution before being kept stirring for 2 hoursunder N₂ atmosphere. After 2 hours the solution was reacted with amixture of bistributylammonium pyrophosphate (TBAPP or (n-Bu₃NH)₂H₂P₂O₇)(652.0 mg, 1.19 mmol, 6.60 equiv.) and tributylamine (175.0 ul, 0.72mmol, 4.0 equiv.) in 1.3 ml of dimethylformamide. After approximately 15minutes, the reaction was quenched with 12.0 ml of 0.2M triethylammoniumbicarbonate (TEAB) and the clear solution was stirred at roomtemperature for an hour. The reaction mixture was lyophilized overnightand the crude reaction mixture was purified by HPLC (Shimadzu, KyotoJapan, Phenomenex C18 preparative column, 250×21.20 mm, 10.0 micron;gradient: 100% A for 3.0 min, then 1% B/min, A=100 mM TEAB buffer,B=ACN; flow rate: 10.0 mL/min; retention time: 18.52-19.57 min).Fractions containing the desired compound were pooled and lyophilized toprovide the NTP of compound 44. The triphosphorylation reactions werecarried out in a two-neck flask flame-dried under N₂ atmosphere.Nucleosides and the protein sponge were dried over P₂O₅ under vacuumovernight prior to use. The formation of monophosphates was monitored byLCMS.

Example 52 Synthesis of N1-methyl pseudouridine (compound 45) andN1-methyl pseudoUTP (NTP of said compound)

A solution of N1-methyl pseudouridine (compound 45) (96.6 mg, 0.374mmol, plus heat to make it soluble) was added to proton sponge (120.0mg, 0.56 mmol, 1.50 equiv.) in 0.8 mL trimethylphosphate (TMP) and wasstirred for 10 minutes at 0° C. Phosphorous oxychloride (POCl₃) (70.0ul, 0.75 mmol, 2.0 equiv.) was added dropwise to the solution beforebeing kept stirring for 2 hours under N₂ atmosphere. After 2 hours thesolution was reacted with a mixture of bistributylammonium pyrophosphate(TBAPP or (n-Bu₃NH)₂H₂P₂O₇) (1.36 g, 2.47 mmol, 6.60 equiv.) andtributylamine (362.0 ul, 1.5 mmol, 4.0 equiv.) in 2.5 ml ofdimethylformamide. After approximately 15 minutes, the reaction wasquenched with 17.0 ml of 0.2M triethylammonium bicarbonate (TEAB) andthe clear solution was stirred at room temperature for an hour. Thereaction mixture was lyophilized overnight and the crude reactionmixture was purified by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18preparative column, 250×21.20 mm, 10.0 micron; gradient: 100% A for 3.0min, then 1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;retention time: 15.91-17.01 min). Fractions containing the desiredcompound were pooled and lyophilized was subjected to atriphosphorylation reaction to provide the NTP of compound 45. Thetriphosphorylation reactions were carried out in a two-neck flaskflame-dried under N₂ atmosphere. Nucleosides and the protein sponge weredried over P₂O₅ under vacuum overnight prior to use. The formation ofmonophosphates was monitored by LCMS.

Example 53 Synthesis of 5-methoxycarbonylethenyl uridine (compound 46)and 5-methoxycarbonylethenyl UTP (NTP of said compound)

A solution of 5-methoxycarbonylethenyl uridine (compound 46) (102.0 mg,0.31 mmol) was added to proton sponge (99.65 mg, 0.46 mmol, 1.50 equiv.)in 0.8 mL trimethylphosphate (TMP) and was stirred for 10 minutes at 0°C. Phosphorous oxychloride (POCl₃) (57.8 ul, 0.62 mmol, 2.0 equiv) wasadded dropwise to the solution before being kept stirring for 2 hoursunder N₂ atmosphere. After 2 hours the solution was reacted with amixture of bistributylammonium pyrophosphate (TBAPP or (n-Bu₃NH)₂H₂P₂O₇)(1.12 g, 2.05 mol, 6.60 equiv.) and tributylamine (300.0 ul, 1.24 mmol,4.0 equiv.) in 2.5 ml of dimethylformamide. After approximately 15minutes, the reaction was quenched with 20.0 ml of 0.2M triethylammoniumbicarbonate (TEAB) and the clear solution was stirred at roomtemperature for an hour. The reaction mixture was lyophilized overnightand the crude reaction mixture was purified by HPLC (Shimadzu, KyotoJapan, Phenomenex C18 preparative column, 250×21.20 mm, 10.0 micron;gradient: 100% A for 3.0 min, then 1% B/min, A=100 mM TEAB buffer,B=ACN; flow rate: 10.0 mL/min; retention time: 21.56-23.21 min).Fractions containing the desired compound were pooled and lyophilized toprovide the NTP of compound 46. The triphosphorylation reactions werecarried out in a two-neck flask flame-dried under N₂ atmosphere.Nucleosides and the protein sponge were dried over P₂O₅ under vacuumovernight prior to use. The formation of monophosphates was monitored byLCMS.

Example 54 Synthesis of 5-aminopropenyl uridine (compound 47) and5-aminopropenyl UTP (NTP of said compound)

5-Aminopropenyl uridine 47 was protected and a solution of protectedcompound 47 (86.0 mg, 0.22 mmol) was added to proton sponge (70.7 mg,0.33 mmol, 1.50 equiv.) in 0.7 mL trimethylphosphate (TMP) and wasstirred for 10 minutes at 0° C. Phosphorous oxychloride (POCl₃) (41.1ul, 0.44 mmol, 2.0 equiv.) was added dropwise to the solution beforebeing kept stirring for 2 hours under N₂ atmosphere. After 2 hours thesolution was reacted with a mixture of bistributylammonium pyrophosphate(TBAPP or (n-Bu₃NH)₂H₂P₂O₇) (784.6 mg, 1.43 mmol, 6.50 equiv.) andtributylamine (213.0 ul, 0.88 mmol, 4.0 equiv.) in 1.6 ml ofdimethylformamide. After approximately 15 minutes, the reaction wasquenched with 15.0 ml of 0.2M triethylammonium bicarbonate (TEAB) andthe clear solution was stirred at room temperature for an hour. 18.0 mlof concentrated ammonium hydroxide was added to the reaction mixture toremove the trifluoroacetyl group. It was then stored stirring overnight.The reaction mixture was lyophilized overnight and the crude reactionmixture was purified by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18preparative column, 250×21.20 mm, 10.0 micron; gradient: 100% A for 3.0min, then 1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;retention time: 16.14-17.02 min). Fractions containing the desiredcompound were pooled and lyophilized to provide the NTP of compound 47.The triphosphorylation reactions were carried out in a two-neck flaskflame-dried under N₂ atmosphere. Nucleosides and the protein sponge weredried over P₂O₅ under vacuum overnight prior to use. The formation ofmonophosphates was monitored by LCMS.

Example 55 Synthesis of N-PEG adenosine (compound 48) and N-PEG ATP (NTPof said compound)

N-PEG adenosine 48 was protected and a solution of the protectedcompound 48 (100.0 mg, 0.15 mmol) was added to proton sponge (49.3 mg,0.23 mmol, 1.50 equiv.) in 0.65 mL trimethylphosphate (TMP) and wasstirred for 10 minutes at 0° C. Phosphorous oxychloride (POCl₃) (28.0ul, 0.3 mmol, 2.0 equiv.) was added dropwise to the solution beforebeing kept stirring for 2 hours under N₂ atmosphere. After 2 hours thesolution was reacted with a mixture of bistributylammonium pyrophosphate(TBAPP or (n-Bu₃NH)₂H₂P₂O₇) (537.7 mg, 0.98 mmol, 6.50 equiv.) andtributylamine (146.0 ul, 0.6 mmol, 4.0 equiv.) in 1.2 ml ofdimethylformamide. After approximately 15 minutes, the reaction wasquenched with 10.0 ml of 0.2M triethylammonium bicarbonate (TEAB) andthe clear solution was stirred at room temperature for an hour. 18.0 mlof concentrated ammonium hydroxide was added to the reaction mixture toremove the trifluoroacetyl group. It was then stored stirring overnight.The reaction mixture was lyophilized overnight and the crude reactionmixture was purified by HPLC (Shimadzu, Kyoto Japan, Phenomenex C18preparative column, 250×21.20 mm, 10.0 micron; gradient: 100% A for 3.0min, then 1% B/min, A=100 mM TEAB buffer, B=ACN; flow rate: 10.0 mL/min;retention time: 24.5-25.5 min). Fractions containing the desiredcompound were pooled and lyophilized to provide the NTP of compound 48.The triphosphorylation reactions were carried out in a two-neck flaskflame-dried under N₂ atmosphere. Nucleosides and the protein sponge weredried over P₂O₅ under vacuum overnight prior to use. The formation ofmonophosphates was monitored by LCMS.

Example 56 Synthesis of N-methyl adenosine (compound 49) and N-methylATP (NTP of said compound)

A solution of N-methyl adenosine (compound 49) (70.0 mg, 0.25 mmol) wasadded to proton sponge (79.29 mg, 0.37 mmol, 1.50 equiv.) in 0.7 mLtrimethylphosphate (TMP) and was stirred for 10 minutes at 0° C.Phosphorous oxychloride (POCl₃) (46.66 ul, 0.50 mmol, 2.0 equiv.) wasadded dropwise to the solution before being kept stirring for 2 hoursunder N₂ atmosphere. After 2 hours the solution was reacted with amixture of bistributylammonium pyrophosphate (TBAPP or (n-Bu₃NH)₂H₂P₂O₇)(888.85 mg, 1.62 mmol, 6.50 equiv.) and tributylamine (241.0 ul, 1.0mmol, 4.0 equiv.) in 1.3 ml of dimethylformamide. After approximately 15minutes, the reaction was quenched with 16.0 ml of 0.2 Mtriethylammonium bicarbonate (TEAB) and the clear solution was stirredat room temperature for an hour. The reaction mixture was lyophilizedovernight and the crude reaction mixture was purified by HPLC (Shimadzu,Kyoto Japan, Phenomenex C18 preparative column, 250×21.20 mm, 10.0micron; gradient: 100% A for 3.0 min, then 1% B/min, A=100 mM TEABbuffer, B=ACN; flow rate: 10.0 mL/min; retention time: 19.62-20.14 min).Fractions containing the desired compound were pooled and lyophilized toprovide the NTP of compound 49. The triphosphorylation reactions werecarried out in a two-neck flask flame-dried under N₂ atmosphere.Nucleosides and the protein sponge were dried over P₂O₅ under vacuumovernight prior to use. The formation of monophosphates was monitored byLCMS.

Example 57 Synthesis of N,N-dimethyl guanosine (compound 50) andN,N-dimethyl GTP (NTP of said compound)

A solution of N,N-dimethyl guanosine (compound 50) (65.8 mg, 0.21 mmol)was added to proton sponge (68.58 mg, 0.32 mmol, 1.50 equiv) in 0.7 mLtrimethylphosphate (TMP) and was stirred for 10 minutes at 0° C.Phosphorous oxychloride (POCl₃) (39.20 ul, 0.42 mmol, 2.0 equiv.) wasadded dropwise to the solution before being kept stirring for 2 hoursunder N₂ atmosphere. After 2 hours the solution was reacted with amixture of bistributylammonium pyrophosphate (TBAPP or (n-Bu₃NH)₂H₂P₂O₇)(751.67 mg, 1.37 mmol, 6.50 equiv.) and tributylamine (204.0 ul, 0.84mmol, 4.0 equiv.) in 1.5 ml of dimethylformamide. After approximately 15minutes, the reaction was quenched with 14.0 ml of 0.2 Mtriethylammonium bicarbonate (TEAB) and the clear solution was stirredat room temperature for an hour. The reaction mixture was lyophilizedovernight and the crude reaction mixture was purified by HPLC (Shimadzu,Kyoto Japan, Phenomenex C18 preparative column, 250×21.20 mm, 10.0micron; gradient: 100% A for 3.0 min, then 1% B/min, A=100 mM TEABbuffer, B=ACN; flow rate: 10.0 mL/min; retention time: 19.27-19.95 min).Fractions containing the desired compound were pooled and lyophilized toprovide the NTP of compound 50. The triphosphorylation reactions werecarried out in a two-neck flask flame-dried under N₂ atmosphere.Nucleosides and the protein sponge were dried over P₂O₅ under vacuumovernight prior to use. The formation of monophosphates was monitored byLCMS.

Example 58 General methods for triphosphate synthesis of NTPS

The nucleoside i can be phosphorylated by any useful method to provide atriphosphate compound 11. For example, the nucleoside can be added toproton sponge and trimethylphosphate (TMP) and cooled (e.g., to −40°C.). Phosphorous oxychloride (POCl₃) can be added dropwise beforereacting with bistributylammonium pyrophosphate (TBAPP or(n-Bu₃NH)₂H₂P₂O₇) and tributylamine. The reaction can then be quicklyquenched with triethylammonium bicarbonate (TEAB). Exemplary conditionsare provided in U.S. Pat. No. 7,893,227, which is incorporated herein byreference.

After the phosphorylation reaction, the reaction mixture can beoptionally lyophilized, purified (e.g., by ion-exchange chromatographyand/or HPLC), or converted to a sodium salt (e.g., by dissolving in MeOHand adding sodium perchlorate in acetone).

Example 59 PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂O dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly-T tracts can be used to adjust the length of the poly-A tail in themRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 60 In vitro Transcription (IVT)

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

Template cDNA 1.0 μg 10x transcription buffer (400 mM Tris-HCl pH 2.0 μl8.0, 190 mM MgCl2, 50 mM DTT, 10 mM Spermidine) Custom NTPs (25 mM each7.2 μl RNase Inhibitor 20 U T7 RNA polymerase 3000 U dH₂0 up to 20.0 μl

-   -   Incubation at 370 C for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

The T7 RNA polymerase may be selected from, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to, the novelpolymerases able to incorporate modified NTPs as well as thosepolymerases described by Liu (Esvelt et al. (Nature (2011) 472(7344):499-503 and U.S. Publication No. 20110177495) which recognizealternate promoters, Ellington (Chelliserrykattil and Ellington, NatureBiotechnology (2004) 22 (9):1155-1160) describing a T7 RNA polymerasevariant to transcribe 2′-O-methyl RNA and Sousa (Padilla and Sousa,Nucleic Acids Research (2002) 30(24): e128) describing a T7 RNApolymerase double mutant; herein incorporated by reference in theirentireties.

Example 61 Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400 U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 62 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the poly-Atailing reaction may not always result in exactly 160 nucleotides. Hencepoly-A tails of approximately 160 nucleotides, e.g, about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 63 Method of Screening for Protein Expression A. ElectrosprayIonization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for electrospray ionization (ESI) using 1, 2, 3 or4 mass analyzers. A biologic sample may also be analyzed using a tandemESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for matrix-assisted laser desorption/ionization(MALDI).

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which may contain proteins encoded by modified RNA,may be treated with a trypsin enzyme to digest the proteins containedwithin. The resulting peptides are analyzed by liquidchromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Thepeptides are fragmented in the mass spectrometer to yield diagnosticpatterns that can be matched to protein sequence databases via computeralgorithms. The digested sample may be diluted to achieve 1 ng or lessstarting material for a given protein. Biological samples containing asimple buffer background (e.g. water or volatile salts) are amenable todirect in-solution digest; more complex backgrounds (e.g. detergent,non-volatile salts, glycerol) require an additional clean-up step tofacilitate the sample analysis.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

Example 64 Cytokine Study: PBMC A. PBMC Isolation and Culture

50 mL of human blood from two donors was received from Research BloodComponents (lots KP30928 and KP30931) in sodium heparin tubes. For eachdonor, the blood was pooled and diluted to 70 mL with DPBS (SAFCBioscience 59331C, lot 071M8408) and split evenly between two 50 mLconical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot10074400) was gently dispensed below the blood layer. The tubes werecentrifuged at 2000 rpm for 30 minutes with low acceleration andbraking. The tubes were removed and the buffy coat PBMC layers weregently transferred to a fresh 50 mL conical and washed with DPBS. Thetubes were centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended andwashed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10minutes. This wash step was repeated, and the PBMC pellets wereresuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) andcounted. The cell suspensions were adjusted to a concentration of3.0×10̂6 cells/mL live cells.

These cells were then plated on five 96 well tissue culture treatedround bottom plates (Costar 3799) per donor at 50 uL per well. Within 30minutes, transfection mixtures were added to each well at a volume of 50uL per well. After 4 hours post transfection, the media was supplementedwith 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368)

B. Transfection Preparation

Modified mRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO: 1;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1) (containing either (1) natural NTPs, (2) 100% substitutionwith 5-methyl cytidine and pseudouridine, or (3) 100% substitution with5-methyl cytidine and N1-methyl pseudouridine; mRNA encoding luciferase(IVT cDNA sequence shown in SEQ ID NO: 2; mRNA sequence shown in SEQ IDNO: 3, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap 1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) (containingeither (1) natural NTPs or (2) 100% substitution with 5-methyl cytidineand pseudouridine) and TLR agonist R⁸⁴⁸ (Invivogen tlrl-r848) werediluted to 38.4 ng/uL in a final volume of 2500 uL Optimem I.

Separately, 110 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot1070962) was diluted with 6.76 mL Optimem I. In a 96 well plate ninealiquots of 135 uL of each mRNA, positive control (R-848) or negativecontrol (Optimem I) was added to 135 uL of the diluted Lipofectamine2000. The plate containing the material to be transfected was incubatedfor 20 minutes. The transfection mixtures were then transferred to eachof the human PBMC plates at 50 uL per well. The plates were thenincubated at 37° C. At 2, 4, 8, 20, and 44 hours each plate was removedfrom the incubator, and the supernatants were frozen.

After the last plate was removed, the supernatants were assayed using ahuman G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit(Thermo Scientific 41105-2). Each condition was done in duplicate.

C. Protein and Innate Immune Response Analysis

The ability of unmodified and modified mRNA to produce the encodedprotein was assessed (G-CSF production) over time as was the ability ofthe mRNA to trigger innate immune recognition as measured byinterferon-alpha production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102).

Results were interpolated against the standard curve of each ELISA plateusing a four parameter logistic curve fit. Shown in Tables 4 and 5 arethe average from 3 separate PBMC donors of the G-CSF, interferon-alpha(IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) production overtime as measured by specific ELISA.

In the G-CSF ELISA, background signal from the Lipofectamine 2000(LF2000) untreated condition was subtracted at each time point. The datademonstrated specific production of human G-CSF protein by humanperipheral blood mononuclear is seen with G-CSF mRNA containing naturalNTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or100% substitution with 5-methyl cytidine and N1-methyl pseudouridine.Production of G-CSF was significantly increased through the use of5-methyl cytidine and N1-methyl pseudouridine modified mRNA relative to5-methyl cytidine and pseudouridine modified mRNA.

With regards to innate immune recognition, while both modified mRNAchemistries largely prevented IFN-alpha and TNF-alpha productionrelative to positive controls (R848, p(I)p(C)), significant differencesdid exist between the chemistries. 5-methyl cytidine and pseudouridinemodified mRNA resulted in low but detectable levels of IFN-alpha andTNF-alpha production, while 5-methyl cytidine and N1-methylpseudouridine modified mRNA resulted in no detectable IFN-alpha andTNF-alpha production.

Consequently, it has been determined that, in addition to the need toreview more than one cytokine marker of the activation of the innateimmune response, it has surprisingly been found that combinations ofmodifications provide differing levels of cellular response (proteinproduction and immune activation). The modification, N1-methylpseudouridine, in this study has been shown to convey added protectionover the standard combination of 5-methylcytidine/pseudouridine exploredby others resulting in twice as much protein and almost 150 foldreduction in immune activation (TNF-alpha).

Given that PBMC contain a large array of innate immune RNA recognitionsensors and are also capable of protein translation, it offers a usefulsystem to test the interdependency of these two pathways. It is knownthat mRNA translation can be negatively affected by activation of suchinnate immune pathways (Kariko et al. Immunity (2005) 23:165-175; Warrenet al. Cell Stem Cell (2010) 7:618-630). Using PBMC as an in vitro assaysystem it is possible to establish a correlation between translation (inthis case G-CSF protein production) and cytokine production (in thiscase exemplified by IFN-alpha and TNF-alpha protein production). Betterprotein production is correlated with lower induction of innate immuneactivation pathway, and new chemistries can be judged favorably based onthis ratio (Table 6).

In this study, the PC Ratio for the two chemical modifications,pseudouridine and N1-methyl pseudouridine, both with 5-methy cytosinewas 4742/141=34 as compared to 9944/1=9944 for the cytokine IFN-alpha.For the cytokine, TNF-alpha, the two chemistries had PC Ratios of 153and 1243, respectively suggesting that for either cytokine, theN1-methylpseudouridine is the superior modification. In Tables 4 and 5,“NT” means not tested.

TABLE 4 G-CSF G-CSF: 3 Donor Average (pg/ml) G-CSF 4742 5-methylcytosine/pseudouridine G-CSF 99445-methylcytosine/N1-methylpseudouridine Luciferase 18 LF2000 16

TABLE 5 IFN-alpha and TNF-alpha IFN-alpha: 3 Donor TNF-alpha: 3 DonorAverage (pg/ml) Average (pg/ml) G-CSF 141 31 5-methyl cytosine/pseudouridine G-CSF 1 8 5-methylcytosine/ N1-methylpseudouridineP(I)P(C) 1104 NT R-848 NT 1477 LF2000 17 25

TABLE 6 G-CSF to Cytokine Ratios G-CSF/IFN-alpha (ratio) G-CSF/TNF-alpha(ratio) 5-methyl- 5-methyl- 5-methyl cytosine/ 5-methyl cytosine/cytosine/ N1-methyl- cytosine/ N1-methyl- pseudo- pseudo- pseudo-pseudo- uridine uridine uridine uridine PC Ratio 34 9944 153 1243

Example 65 Chemical Modification Ranges of Modified mRNA

Modified nucleosides such as, but not limited to, the chemicalmodifications 5-methylcytosine and pseudouridine have been shown tolower the innate immune response and increase expression of RNA inmammalian cells. Surprisingly and not previously known, the effectsmanifested by these chemical modifications can be titrated when theamount of chemical modification of a particular nucleotide is less than100%. Previously, it was believed that the benefit of chemicalmodification could be derived using less than complete replacement of amodified nucleoside and published reports suggest no loss of benefituntil the level of substitution with a modified nucleoside is less than50% (Kariko et al., Immunity (2005) 23:165-175).

However, it has now been shown that the benefits of chemicalmodification are directly correlated with the degree of chemicalmodification and must be considered in view of more than a singlemeasure of immune response. Such benefits include enhanced proteinproduction or mRNA translation and reduced or avoidance of stimulatingthe innate immune response as measured by cytokine profiles and metricsof immune response triggers.

Enhanced mRNA translation and reduced or lack of innate immunestimulation are seen with 100% substitution with a modified nucleoside.Lesser percentages of substitution result in less mRNA translation andmore innate immune stimulation, with unmodified mRNA showing the lowesttranslation and the highest innate immune stimulation.

In Vitro PBMC Studies: Percent modification

480 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA(SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) was completely modified with 5mC and pseudo(100% modification), not modified with 5mC and pseudo (0% modification)or was partially modified with 5mC and pseudoU so the mRNA would contain75% modification, 50% modification or 25% modification. A control sampleof Luciferase (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1;fully modified 5meC and pseudoU) was also analyzed for G-CSF expression.For TNF-alpha and IFN-alpha control samples of Lipofectamine-2000, LPS,R-848, Luciferase (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1;fully modified 5mC and pseudo), and P(I)P(C) were also analyzed. Thesupernatant was harvested and run by ELISA 22 hours after transfectionto determine the protein expression. The expression of G-CSF is shown inTable 7 and the expression of IFN-alpha and TNF-alpha is shown in Table8. The expression of IFN-alpha and TNF-alpha may be a secondary effectfrom the transfection of the G-CSF mRNA. Tables 7, 8 and FIG. 10 showthat the amount of chemical modification of G-CSF, interferon alpha(IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) is titratablewhen the mRNA is not fully modified and the titratable trend is not thesame for each target.

As mentioned above, using PBMC as an in vitro assay system it ispossible to establish a correlation between translation (in this caseG-CSF protein production) and cytokine production (in this caseexemplified by IFN-alpha protein production). Better protein productionis correlated with lower induction of innate immune activation pathway,and the percentage modification of a chemistry can be judged favorablybased on this ratio (Table 9). As calculated from Tables 7 and 8 andshown in Table 9, full modification with 5-methylcytidine andpseudouridine shows a much better ratio of protein/cytokine productionthan without any modification (natural G-CSF mRNA) (100-fold forIFN-alpha and 27-fold for TNF-alpha). Partial modification shows alinear relationship with increasingly less modification resulting in alower protein/cytokine ratio.

TABLE 1 G-CSF Expression G-CSF Expression (pg/ml) D1 D2 D3 100%modification  1968.9 2595.6 2835.7 75% modification 566.7 631.4 659.550% modification 188.9 187.2 191.9 25% modification 139.3 126.9 102.0 0% modification 194.8 182.0 183.3 Luciferase 90.2 0.0 22.1

TABLE 8 IFN-alpha and TNF-alpha Expression IFN-alpha TNF-alphaExpression (pg/ml) Expression (pg/ml) D1 D2 D3 D1 D2 D3 100%modification 336.5 78.0 46.4 115.0 15.0 11.1 75% modification 339.6107.6 160.9 107.4 21.7 11.8 50% modification 478.9 261.1 389.7 49.6 24.110.4 25% modification 564.3 400.4 670.7 85.6 26.6 19.8 0% modification1421.6 810.5 1260.5 154.6 96.8 45.9 LPS 0.0 0.6 0.0 0.0 12.6 4.3 R-8480.5 3.0 14.1 655.2 989.9 420.4 P(I)P(C) 130.8 297.1 585.2 765.8 2362.71874.4 Lipid only 1952.2 866.6 855.8 248.5 82.0 60.7

TABLE 9 PC Ratio and Effect of Percentage of Modification AverageAverage Average G-CSF/IFN- G-CSF/TNF- % G-CSF IFN-a TNF-a alpha alphaModification (pg/ml) (pg/ml) (pg/ml) (PC ratio) (PC ratio) 100 2466 15347 16 52 75 619 202 47 3.1 13 50 189 376 28 0.5 6.8 25 122 545 44 0.22.8 0 186 1164 99 0.16 1.9

Example 66 Modified RNA transfected in PBMC

500 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA(SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) was completely modified with 5mC and pseudo(100% modification), not modified with 5mC and pseudo (0% modification)or was partially modified with 5mC and pseudoU so the mRNA would contain50% modification, 25% modification, 10% modification, %5 modification,1% modification or 0.1% modification. A control sample of mCherry (mRNAsequence shown in SEQ ID NO: 6; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified 5meC andpseudouridine) and G-CSF fully modified with 5-methylcytosine andpseudouridine (Control G-CSF) was also analyzed for G-CSF expression.For tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha(IFN-alpha) control samples of Lipofectamine-2000, LPS, R-848,Luciferase (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified 5mC and pseudo), and P(I)P(C) were also analyzed. Thesupernatant was harvested 6 hours and 18 hours after transfection andrun by ELISA to determine the protein expression. The expression ofG-CSF, IFN-alpha, and TNF-alpha for Donor 1 is shown in Table 10, Donor2 is shown in Table 11 and Donor 3 is shown in Table 12.

Full 100% modification with 5-methylcytidine and pseudouridine resultedin the most protein translation (G-CSF) and the least amount of cytokineproduced across all three human PBMC donors. Decreasing amounts ofmodification results in more cytokine production (IFN-alpha andTNF-alpha), thus further highlighting the importance of fullymodification to reduce cytokines and to improve protein translation (asevidenced here by G-CSF production).

TABLE 10 Donor 1 IFN-alpha TNF-alpha G-CSF (pg/mL) (pg/mL) (pg/mL) 6 186 18 6 18 hours hours hours hours hours hours 100% Mod 1815 2224 1 13 00 75% Mod 591 614 0 89 0 0 50% Mod 172 147 0 193 0 0 25% Mod 111 92 2219 0 0 10% Mod 138 138 7 536 18 0 1% Mod 199 214 9 660 18 3 0.1% Mod222 208 10 597 0 6 0% Mod 273 299 10 501 10 0 Control G-CSF 957 1274 3123 18633 1620 mCherry 0 0 0 10 0 0 Untreated N/A N/A 0 0 1 1

TABLE 11 Donor 2 IFN-alpha TNF-alpha G-CSF (pg/mL) (pg/mL) (pg/mL) 6 186 18 6 18 hours hours hours hours hours hours 100% Mod 2184 2432 0 7 011 75% Mod 935 958 3 130 0 0 50% Mod 192 253 2 625 7 23 25% Mod 153 1587 464 6 6 10% Mod 203 223 25 700 22 39 1% Mod 288 275 27 962 51 66 0.1%Mod 318 288 33 635 28 5 0% Mod 389 413 26 748 1 253 Control G-CSF 14611634 1 59 481 814 mCherry 0 7 0 1 0 0 Untreated N/A N/A 1 0 0 0

TABLE 12 Donor 3 IFN-alpha TNF-alpha G-CSF (pg/mL) (pg/mL) (pg/mL) 6 186 18 6 18 hours hours hours hours hours hours 100% Mod 6086 7549 7 65811 11 75% Mod 2479 2378 23 752 4 35 50% Mod 667 774 24 896 22 18 25% Mod480 541 57 1557 43 115 10% Mod 838 956 159 2755 144 123 1% Mod 1108 1197235 3415 88 270 0.1% Mod 1338 1177 191 2873 37 363 0% Mod 1463 1666 2153793 74 429 Control G-CSF 3272 3603 16 1557 731 9066 mCherry 0 0 2 645 00 Untreated N/A N/A 1 1 0 8

Example 67 Microames Reverse Mutation Screen of Modifications Backgroundand Methods

The microames screen is a version of the full Ames preincubation assay.It detects both frameshift and base-pair substitution mutations usingfour Salmonella tester strains (TA97a, TA98, TA100 and TA1535) and oneEscherichia coli strain (WP2 uvrA pKM101). Strains TA97a and TA98 detectframeshift mutations, and TA100, TA1535 and WP2 uvrA pKM101 detectbase-pair substitution mutations. This scaled-down Ames test usesminimal compound, is conducted with and without metabolic activation (S9fraction), and uses multiwell plates. This teste is a microbial assay todetect the mutagenic potential of test compounds.

The microAmes screen for 5-Methylcytidine, Pseudouridine orN′-methylpseudouridine test article was tested in duplicate with strainsTA97a, TA98, TA100, TA1535 and WP2 uvrA pKM101 in the presence andabsence of a metabolic activation system (AROCLOR™ 1254 induced ratliver S9 microsomal fraction) at 0.25, 2.5, 12.5, 25, 75, and 250ug/well. Positive control compounds were used at 4 differentconcentrations to ensure the assay system was sensitive to knownmutagenic compounds. DMSO was used as the vehicle control. Positive andvehicle controls yielded the expected results, demonstrating that themicroAmes screen is sufficiently sensitive to detect mutagens.

Results

For 5-methylcytosine, precipitates were not observed with any testerstrain either with or without metabolic activation. Cytotoxicity(reduction in the background lawn and/or number of revertants) was notobserved in any strain either with or without metabolic activation.There was no increase in the number of revertant colonies as comparedwith the vehicle control in any strain with or without metabolicactivation. Therefore, 5-Methylcytidine was not mutagenic up to 250ug/well in strains TA97a, TA98, TA100, TA1535 and WP2 uvrA pKM101 withor without metabolic activation under the conditions of the microAmesscreen.

Precipitates were not observed with any tester strain either with orwithout metabolic activation for pseudouridine. Cytotoxicity (reductionin the number of revertants) was observed with strain TA100 withoutmetabolic activation. Cytotoxicity (reduction in the background lawnand/or number of revertants) was not observed in any other strain eitherwith or without metabolic activation. There was no increase in thenumber of revertant colonies as compared with the vehicle control in anystrain with or without metabolic activation. Therefore, pseudouridinewas not mutagenic up to 75 ug/well in strain TA100 without metabolicactivation and up to 250 μg/well in strains TA97a, TA98, TA1535 and WP2uvrA pKM101 with or without metabolic activation and strain TA100without metabolic activation under the conditions of this microAmesscreen.

For the modification, N1-methylpseudouridine precipitates were notobserved with any tester strain either with or without metabolicactivation. Cytotoxicity (reduction in the background lawn and/or numberof revertants) was not observed in any strain either with or withoutmetabolic activation. There was no increase in the number of revertantcolonies as compared with the vehicle control in any strain with orwithout metabolic activation. N1-methylpseudouridine was not mutagenicup to 250 μg/well in strains TA97a, TA98, TA100, TA1535 and WP2 uvrApKM101 with or without metabolic activation under the conditions of thismicroAmes screen. N1-methylpseudouridine was found less mutagenic thanpseudouridine.

The comparison in this microAMES test of 5 methyl cytidine,pseudouridine, and N1-methylpseudouridine reveal them to be generallynon-mutagenic. Of particular note, however, was the difference betweenpseudouridine and N1-methylpseudouridine, where pseudouridine did show acytotoxic response in one bacterial strain where N1-methylpseudouridinedid not. These microAMES tests are routinely used as part of thepre-clinical assessment of compound safety and highlight an importantdifference between N1-methylpseudouridine and pseudouridine.

Example 68 Toxicity of Nucleoside Triphosphates (NTPs)

The cytotoxicity of natural and modified nucleoside triphosphates (NTPs)alone or in combination with other bases, was analyzed in humanembryonic kidney 293 (HEK293) cells in the absence of transfectionreagent. HEK293 cells were seeded on 96-well plates at a density of30,000 cells per well having 0.75 ul of RNAiMAX™ (Invitrogen, Carlsbad,Calif.) per well at a total well volume of 100 ul. 10 ul of the NTPsoutlined in Table 12 were combined with 10 ul of lipid dilution andincubated for 30 minutes to form a complex before 80 ul of the HEK293cell suspension was added to the NTP complex.

Natural and modified NTPs were transfected at a concentration of 2.1 nM,21 nM, 210 nM, 2.1 um, 21 uM, 210 um or 2.1 mM. NTPs in combination weretransfected at a total concentration of NTPs of 8.4 nM, 84 nM, 840 nM,8.4 uM, 84 uM, 840 uM and 8.4 mM. As a control modified G-CSF mRNA (SEQID NO: 1; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap 1; fully modified 5-methylcytosine andpseudouridine) was transfected in HEK293 cells at a concentration of 8.4nM. The cytotoxicity of the NTPs and the modified G-CSF mRNA was assayedat 4, 24, 48 and 72 hours post addition to the HEK293 cells using a CYTOTOX-GLO™ assay from Promega (Madison, Wis.) following the manufacturerprotocol except pippeting was used for lysing the cells instead ofshaking the plates.

Table 13 and 14 show the percent of viable cells for each of the NTPs,NTP combinations and controls tested. There was no toxicity seen withthe individual NTPs as compared to the untreated cells. These datademonstrate that introduction of individual NTPs, including5-methylcytidine, pseudouridine, and N1-methylpseudouridine, intomammalian cells is not toxic at doses 1,000,000 times an effective dosewhen introduced as a modified mRNA.

TABLE 13 Cytotoxicity of Individual NTPs Individual NTP CytotoxicityDose Time 2.1 mM 210 uM 21 uM 2.1 uM 210 nM 21 nM 2.1 nM Adenine  4 hr90.03 85.97 91.20 90.23 90.36 93.21 93.48 24 hr 88.42 87.31 86.86 86.8186.94 87.19 86.44 48 hr 93.71 90.55 89.94 89.80 89.17 91.13 92.12 72 hr97.49 94.81 93.83 94.58 92.22 93.88 95.74 Cytosine  4 hr 90.51 89.8891.41 90.49 88.95 93.11 93.34 24 hr 86.92 86.33 85.72 86.70 86.12 86.1685.78 48 hr 94.23 87.81 87.28 87.73 85.36 88.95 88.99 72 hr 97.15 92.3492.22 88.93 88.22 91.80 94.22 Guanine  4 hr 90.96 90.14 91.36 90.6090.00 92.84 93.33 24 hr 86.37 85.86 85.93 86.13 86.35 85.50 85.41 48 hr93.83 87.05 88.18 87.89 85.31 87.92 89.57 72 hr 97.04 91.41 92.39 92.3092.19 92.55 93.72 Uracil  4 hr 90.97 89.60 91.95 90.90 91.05 92.90 93.1524 hr 87.68 86.48 85.89 86.75 86.52 87.23 87.63 48 hr 94.39 88.98 89.1189.44 88.33 88.89 91.28 72 hr 96.82 93.45 93.63 94.60 94.50 94.53 95.51Pseudouridine  4 hr 92.09 92.37 91.35 92.02 92.84 91.96 92.26 24 hr88.38 86.68 86.05 86.75 85.91 87.59 87.31 48 hr 88.62 87.79 87.73 87.6687.82 89.03 91.99 72 hr 96.87 89.82 94.23 93.54 92.37 94.26 94.255-methyl  4 hr 92.01 91.54 91.16 91.31 92.31 91.40 92.23 cytosine 24 hr87.97 85.76 84.72 85.14 84.71 86.37 86.35 48 hr 87.29 85.94 85.74 86.1886.44 87.10 88.18 72 hr 96.08 88.10 92.26 90.92 89.97 92.10 91.93N1-methyl  4 hr 92.45 91.43 91.48 90.41 92.15 91.44 91.89 pseudouridine24 hr 88.92 86.48 85.17 85.72 85.89 86.85 87.79 48 hr 89.84 86.02 87.5285.85 87.38 86.72 87.81 72 hr 96.80 93.03 93.83 92.25 92.40 92.84 92.98Untreated  4 hr 92.77 — — — — — — 24 hr 87.52 — — — — — — 48 hr 92.95 —— — — — — 72 hr 96.97 — — — — — —

TABLE 14 Cytotoxicity of NTPs in Combination NTP CombinationCytotoxicity Dose Time 8.4 mM 840 uM 84 uM 8.4 uM 840 nM 84 nM 8.4 nMPseudouridine/5-  4 hr 92.27 92.04 91.47 90.86 90.87 91.10 91.50methylcytosine/ 24 hr 88.51 86.90 86.43 88.15 88.46 86.28 87.51Adenine/Guanine 48 hr 88.30 87.36 88.58 88.13 87.39 88.72 90.55 72 hr96.53 94.42 94.31 94.53 94.38 94.36 93.65 N1-methyl  4 hr 92.31 91.7191.36 91.15 91.30 90.86 91.38 pseudouridine/5- 24 hr 88.19 87.07 86.4687.70 88.13 85.30 87.21 methylcytosine/ 48 hr 87.17 86.53 87.51 85.8584.69 87.73 86.79 Adenine/Guanine 72 hr 96.40 94.88 94.40 93.65 94.8292.72 93.10 G-CSF  4 hr na na na na na na 92.63 modified 24 hr na na nana na na 87.53 mRNA 48 hr na na na na na na 91.70 72 hr na na na na nana 96.36

Example 69 Innate Immune Response Study in BJ Fibroblasts

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplementedwith 10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts wereseeded on a 24-well plate at a density of 300,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap 1) fully modified with 5-methylcytosine andpseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methylpseudouridine (Gen2) having Cap0, Cap1 or no cap wastransfected using Lipofectamine 2000 (Invitrogen, catalog #11668-019),following manufacturer's protocol. Control samples of poly I:C (PIC),Lipofectamine 2000 (Lipo), natural luciferase mRNA (mRNA sequence shownin SEQ ID NO: 3; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) and natural G-CSF mRNA were also transfected.The cells were harvested after 18 hours, the total RNA was isolated andDNASE® treated using the RNeasy micro kit (catalog #74004) following themanufacturer's protocol. 100 ng of total RNA was used for cDNA synthesisusing High Capacity cDNA Reverse Transcription kit (catalog #4368814)following the manufacturer's protocol. The cDNA was then analyzed forthe expression of innate immune response genes by quantitative real timePCR using SybrGreen in a Biorad CFX 384 instrument followingmanufacturer's protocol. Table 15 shows the expression level of innateimmune response transcripts relative to house-keeping gene HPRT(hypoxanthine phosphoribosytransferase) and is expressed asfold-induction relative to HPRT. In the table, the panel of standardmetrics includes: RIG-I is retinoic acid inducible gene 1, IL6 isinterleukin-6, OAS-1 is oligoadenylate synthetase 1, IFNb isinterferon-beta, AIM2 is absent in melanoma-2, IFIT-1 isinterferon-induced protein with tetratricopeptide repeats 1, PKR isprotein kinase R, TNFa is tumor necrosis factor alpha and IFNa isinterferon alpha.

TABLE 15 Innate Immune Response Transcript Levels Formulation RIG-I IL6OAS-1 IFNb AIM2 IFIT-1 PKR TNFa IFNa Natural 71.5 20.6 20.778 11.4040.251 151.218 16.001 0.526 0.067 Luciferase Natural G-CSF 73.3 47.119.359 13.615 0.264 142.011 11.667 1.185 0.153 PIC 30.0 2.8 8.628 1.5230.100 71.914 10.326 0.264 0.063 G-CSF Gen1-UC 0.81 0.22 0.080 0.0090.008 2.220 1.592 0.090 0.027 G-CSF Gen1-Cap0 0.54 0.26 0.042 0.0050.008 1.314 1.568 0.088 0.038 G-CSF Gen1-Cap1 0.58 0.30 0.035 0.0070.006 1.510 1.371 0.090 0.040 G-CSF Gen2-UC 0.21 0.20 0.002 0.007 0.0070.603 0.969 0.129 0.005 G-CSF Gen2-Cap0 0.23 0.21 0.002 0.0014 0.0070.648 1.547 0.121 0.035 G-CSF Gen2-Cap1 0.27 0.26 0.011 0.004 0.0050.678 1.557 0.099 0.037 Lipo 0.27 0.53 0.001 0 0.007 0.954 1.536 0.1580.064

Example 70 In Vivo Detection of Innate Immune Response

In an effort to distringuish the importance of different chemicalmodification of mRNA on in vivo protein production and cytokine responsein vivo, female BALB/C mice (n=5) are injected intramuscularly withG-CSF mRNA (GCSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 1; polyAtail of approximately 160 nucleotides not shown in sequence;) with a 5′cap of Cap1, G-CSF mRNA fully modified with 5-methylcytosine andpseudouridine (GCSF mRNA 5 mc/pU), G-CSF mRNA fully modified with5-methylcytosine and N1-methylpseudouridine with (GCSF mRNA 5 mc/N1pU)or without a 5′ cap (GCSF mRNA 5 mc/N1 pU no cap) or a control of eitherR848 or 5% sucrose as described in Table 16.

TABLE 16 Dosing Chart Formulation Route Dose (ug/mouse) Dose (ul) GCSFmRNA unmod I.M. 200 50 GCSF mRNA 5 mc/pU I.M. 200 50 GCSF mRNA I.M. 20050 5 mc/N1pU GCSF mRNA I.M. 200 50 5 mc/N1pU no cap R848 I.M. 75 50 5%sucrose I.M. — 50 Untreated I.M. — —

Blood is collected at 8 hours after dosing. Using ELISA the proteinlevels of G-CSF, TNF-alpha and IFN-alpha is determined by ELISA. 8 hoursafter dosing, muscle is collected from the injection site andquantitative real time polymerase chain reaction (QPCR) is used todetermine the mRNA levels of RIG-I, PKR, AIM-2, IFIT-1, OAS-2, MDA-5,IFN-beta, TNF-alpha, IL-6, G-CSF, CD45 in the muscle.

Example 71 In Vivo Detection of Innate Immune Response Study

Female BALB/C mice (n=5) were injected intramuscularly with G-CSF mRNA(GCSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence;) with a 5′ cap ofCap1, G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine(GCSF mRNA 5 mc/pU), G-CSF mRNA fully modified with 5-methylcytosine andN1-methylpseudouridine with (GCSF mRNA 5 mc/N1pU) or without a 5′ cap(GCSF mRNA 5 mc/N1 pU no cap) or a control of either R848 or 5% sucroseas described in Table 17. Blood is collected at 8 hours after dosing andusing ELISA the protein levels of G-CSF and interferon-alpha (IFN-alpha)is determined by ELISA and are shown in Table 17.

As shown in Table 17, unmodified, 5 mc/pU, and 5 mc/N1pU modified G-CSFmRNA resulted in human G-CSF expression in mouse serum. The uncapped5mC/N1pU modified G-CSF mRNA showed no human G-CSF expression in serum,highlighting the importance of having a 5′ cap structure for proteintranslation.

As expected, no human G-CSF protein was expressed in the R848, 5%sucrose only, and untreated groups. Importantly, significant differenceswere seen in cytokine production as measured by mouse IFN-alpha in theserum. As expected, unmodified G-CSF mRNA demonstrated a robust cytokineresponse in vivo (greater than the R848 positive control). The 5 mc/pUmodified G-CSF mRNA did show a low but detectable cytokine response invivo, while the 5 mc/N1pU modified mRNA showed no detectable IFN-alphain the serum (and same as vehicle or untreated animals).

Also, the response of 5 mc/N1pU modified mRNA was the same regardless ofwhether it was capped or not. These in vivo results reinforce theconclusion that 1) that unmodified mRNA produce a robust innate immuneresponse, 2) that this is reduced, but not abolished, through 100%incorporation of 5 mc/pU modification, and 3) that incorporation of 5mc/N1pU modifications results in no detectable cytokine response.

Lastly, given that these injections are in 5% sucrose (which has noeffect by itself), these result should accurately reflect theimmunostimulatory potential of these modifications.

From the data it is evident that N1pU modified molecules produce moreprotein while concomitantly having little or no effect on IFN-alphaexpression. It is also evident that capping is required for proteinproduction for this chemical modification. The Protein: Cytokine Ratioof 748 as compared to the PC Ratio for the unmodified mRNA (PC=9) meansthat this chemical modification is far superior as related to theeffects or biological implications associated with IFN-alpha.

TABLE 17 Human G-CSF and Mouse IFN-alpha in serum Dose G-CSF IFN-alpha(ug/ Dose protein expression PC Formulation Route mouse) (ul) (pg/ml)(pg/ml) Ratio GCSF mRNA I.M. 200 50 605.6 67.01 9 unmod GCSF mRNA I.M.200 50 356.5 8.87 40 5 mc/pU GCSF mRNA I.M. 200 50 748.1 0 748 5 mc/N1pUGCSF mRNA I.M. 200 50 6.5 0 6.5 5 mc/N1pU no cap R848 I.M.  75 50 3.440.97 .08 5% sucrose I.M. — 50 0 1.49 0 Untreated I.M. — — 0 0 0

Example 72 In Vivo Delivery Using Lipoplexes A. Human G-CSF Modified RNA

A formulation containing 100 μg of one of two versions of modified humanG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)(G-CSF fully modified with 5-methylcytosine and pseudouridine (G-CSF) orG-CSF fully modified with 5-methylcytosine and N1-methyl-pseudouridine(G-CSF-N1) lipoplexed with 30% by volume of RNAIMAX™ and delivered in150 uL intramuscularly (I.M.) and in 225 uL intravenously (I.V.) toC57/BL6 mice.

Three control groups were administered either 100 μg of modifiedluciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 2; mRNA sequenceshown in SEQ ID NO: 3, polyA tail of approximately 160 nucleotides notshown in sequence, 5′ cap, Cap 1, fully modified with 5-methylcytosineat each cytosine and pseudouridine replacement at each uridine site)intramuscularly (Luc-unsp I.M.) or 150 μg of modified luciferase mRNAintravenously (Luc-unsp I.V.) or 150 uL of the formulation bufferintramuscularly (Buffer I.M.). 6 hours after administration of aformulation, serum was collected to measure the amount of human G-CSFprotein in the mouse serum by human G-CSF ELISA and the results areshown in Table 18.

These results demonstrate that both 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.V. or I.M. route of administration in a lipoplexformulation.

TABLE 18 Human G-CSF in Serum (I.M. and I.V. Injection Route)Formulation Route G-CSF (pg/ml) G-CSF I.M. 85.6 G-CSF-N1 I.M. 40.1 G-CSFI.V. 31.0 G-CSF-N1 I.V. 6.1 Luc-unsp I.M. 0.0 Luc-unsp I.V. 0.0 BufferI.M. 0.0

B. Human G-CSF Modified RNA Comparison

A formulation containing 100 μg of either modified human G-CSF mRNAlipoplexed with 30% by volume of RNAIMAX™ with a 5-methylcytosine (5 mc)and a pseudouridine (ψ) modification (G-CSF-Gen1-Lipoplex), modifiedhuman G-CSF mRNA with a 5 mc and ψ modification in saline(G-CSF-Gen1-Saline), modified human G-CSF mRNA with aN1-5-methylcytosine (N-1-5 mc) and a ψ modification lipoplexed with 30%by volume of RNAIMAX™ (G-CSF-Gen2-Lipoplex), modified human G-CSF mRNAwith a N1-5 mc and ψ modification in saline (G-CSF-Gen2-Saline),modified luciferase with a 5 mc and ψ modification lipoplexed with 30%by volume of RNAIMAX™ (Luc-Lipoplex), or luciferase mRNA fully modifiedwith 5 mc and ψ modifications in saline (Luc-Saline) was deliveredintramuscularly (I.M.) or subcutaneously (S.C.) and a control group foreach method of administration was giving a dose of 80 uL of theformulation buffer (F. Buffer) to C57/BL6 mice. 13 hours post injectionserum and tissue from the site of injection were collected from eachmouse and analyzed by G-CSF ELISA to compare human G-CSF protein levels.The results of the human G-CSF protein in mouse serum from theintramuscular administration and the subcutaneous administration resultsare shown in Table 19.

These results demonstrate that 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.M. or S.C. route of administration whether in a salineformulation or in a lipoplex formulation. As shown in Table 19,5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNAgenerally demonstrates increased human G-CSF protein production relativeto 5-methylcytosine/pseudouridine modified human G-CSF mRNA.

TABLE 19 Human G-CSF Protein in Mouse Serum G-CSF (pg/ml) FormulationI.M. Injection Route S.C. Injection Route G-CSF-Gen1-Lipoplex 13.98842.855 GCSF-Gen1-saline 9.375 4.614 GCSF-Gen2-lipoplex 75.572 32.107GCSF-Gen2-saline 20.190 45.024 Luc lipoplex 0 3.754 Luc saline 0.0748 0F. Buffer 4.977 2.156

Example 73 Multi-Site Administration: Intramuscular and Subcutaneous

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 1; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap 1) modified as either Gen1 or Gen2 (5-methylcytosine (5 mc) and apseudouridine (ψ) modification, G-CSF-Gen1; or N1-5-methylcytosine(N-1-5 mc) and a ψ modification, G-CSF-Gen2) and formulated in salinewere delivered to mice via intramuscular (IM) or subcutaneous (SC)injection. Injection of four doses or 2×50 ug (two sites) daily forthree days (24 hrs interval) was performed. The fourth dose wasadministered 6 hrs before blood collection and CBC analysis. Controlsincluded Luciferase (cDNA sequence for IVT shown in SEQ ID NO: 2; mRNAsequence shown in SEQ ID NO: 3, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap 1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) or the formulation buffer (F.Buffer). The mice were bledat 72 hours after the first mRNA injection (6 hours after the last mRNAdose) to determine the effect of mRNA-encoded human G-CSF on theneutrophil count. The dosing regimen is shown in Table 20 as are theresulting neutrophil counts (thousands/uL). In Table 20, an asterisks(*) indicate statistical significance at p<0.05.

For intramuscular administration, the data reveal a four fold increasein neutrophil count above control at day 3 for the Gen1 G-CSF mRNA and atwo fold increase for the Gen2 G-CSF mRNA. For subcutaneousadministration, the data reveal a two fold increase in neutrophil countabove control at day 3 for the Gen2 G-CSF mRNA.

These data demonstrate that both 5-methylcytidine/pseudouridine and5-methylcytidine/N1-methylpseudouridine-modified mRNA can bebiologically active, as evidenced by specific increases in bloodneutrophil counts.

TABLE 20 Dosing Regimen Dose Vol. Dosing Neutrophil Gr. Treatment RouteN = Dose (μg/mouse) (μl/mouse) Vehicle Thous/uL 1 G-CSF (Gen1) I.M 5 2 ×50 ug (four doses) 50 F. buffer  840* 2 G-CSF (Gen1) S.C 5 2 × 50 ug(four doses) 50 F. buffer 430 3 G-CSF (Gen2) I.M 5 2 × 50 ug (fourdoses) 50 F. buffer  746* 4 G-CSF (Gen2) S.C 5 2 × 50 ug (four doses) 50F. buffer 683 5 Luc (Gen1) I.M. 5 2 × 50 ug (four doses) 50 F. buffer201 6 Luc (Gen1) S.C. 5 2 × 50 ug (four doses) 50 F. buffer 307 7 Luc(Gen2) I.M 5 2 × 50 ug (four doses) 50 F. buffer 336 8 Luc (Gen2) S.C 52 × 50 ug (four doses) 50 F. buffer 357 9 F. Buffer I.M 4 0 (four doses)50 F. buffer 245 10 F. Buffer S.C. 4 0 (four doses) 50 F. buffer 509 11Untreated — 4 — 312

Example 74 Intravenous Administration

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 1; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap 1) modified with 5-methylcytosine (5 mc) and a pseudouridine (ψ)modification (Gen1); or having no modifications and formulated in 10%lipoplex (RNAIMAX™) were delivered to mice at a dose of 50 ug RNA and ina volume of 100 ul via intravenous (IV) injection at days 0, 2 and 4.Neutrophils were measured at days 1, 5 and 8. Controls includednon-specific mammalian RNA or the formulation buffer alone (F.Buffer).The mice were bled at days 1, 5 and 8 to determine the effect ofmRNA-encoded human G-CSF to increase neutrophil count. The dosingregimen is shown in Table 21 as are the resulting neutrophil counts(thousands/uL; K/uL).

For intravenous administration, the data reveal a four to five foldincrease in neutrophil count above control at day 5 with G-CSF modifiedmRNA but not with unmodified G-CSF mRNA or non-specific controls. Bloodcount returned to baseline four days after the final injection. No otherchanges in leukocyte populations were observed.

In Table 21, an asterisk (*) indicates statistical significance atp<0.001 compared to buffer.

These data demonstrate that lipoplex-formulated5-methylcytidine/pseudouridine-modified mRNA can be biologically active,when delivered through an I.V. route of administration as evidenced byspecific increases in blood neutrophil counts. No other cell subsetswere significantly altered. Unmodified G-CSF mRNA similarly administeredshowed no pharmacologic effect on neutrophil counts.

TABLE 21 Dosing Regimen Dose Vol. Dosing Neutrophil Gr. Treatment N(μl/mouse) Vehicle K/uL 1 G-CSF (Gen1) Day 1 5 100 10% lipoplex 2.91 2G-CSF (Gen1) Day 5 5 100 10% lipoplex 5.32* 3 G-CSF (Gen1) Day 8 5 10010% lipoplex 2.06 4 G-CSF (no 5 100 10% lipoplex 1.88 modification) Day1 5 G-CSF (no 5 100 10% lipoplex 1.95 modification) Day 5 6 G-CSF (no 5100 10% lipoplex 2.09 modification) Day 8 7 RNA control Day 1 5 100 10%lipoplex 2.90 8 RNA control Day 5 5 100 10% lipoplex 1.68 9 RNA controlDay 8 4 100 10% lipoplex 1.72 10 F. Buffer Day 1 4 100 10% lipoplex 2.5111 F. Buffer Day 5 4 100 10% lipoplex 1.31 12 F. Buffer Day 8 4 100 10%lipoplex 1.92

Example 75 Routes of Administration

Studies were performed to investigate split dosing using differentroutes of administration. Studies utilizing multiple subcutaneous orintramuscular injection sites at one time point were designed andperformed to investigate ways to increase modified mRNA drug exposureand improve protein production. In addition to detection of theexpressed protein product, an assessment of the physiological functionof proteins was also determined through analyzing samples from theanimal tested.

Surprisingly, it has been determined that split dosing of modified mRNAproduces greater protein production and phenotypic responses than thoseproduced by single unit dosing or multi-dosing schemes.

The design of a split dose experiment involved using humanerythropoietin (EPO) modified mRNA (mRNA sequence shown in SEQ ID NO: 5;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1) or luciferase modified mRNA (mRNA sequence shown in SEQ IDNO: 3; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap 1) administered in buffer alone or formulated with30% lipoplex (RNAIMAX™). The dosing vehicle (buffer) consisted of 150 mMNaCl, 2 mM CaCl₂, 2 mM Na⁺-phosphate (1.4 mM monobasic sodium phosphate;0.6 mM dibasic sodium phosphate), and 0.5 mM EDTA, pH 6.5. The pH wasadjusted using sodium hydroxide and the final solution was filtersterilized. The mRNA was modified with 5-methylC (5meC) at each cytosineand pseudouridine replacement at each uridine site.

4 mice per group were dosed intramuscularly (I.M.), intravenously (I.V.)or subcutaneously (S.C.) by the dosing chart outlined in Table 22. Serumwas collected 13 hours post injection from all mice, tissue wascollected from the site of injection from the intramuscular andsubcutaneous group and the spleen, liver and kidneys were collected fromthe intravenous group. The results from the intramuscular group and thesubcutaneous group results are shown in Table 23.

TABLE 22 Dosing Chart Total Dosing Group Treatment Route Dose ofmodified mRNA Dose Vehicle 1 Lipoplex-human EPO I.M. 4 × 100 ug + 30%Lipoplex 4 × 70 ul Lipoplex modified mRNA 2 Lipoplex-human EPO I.M. 4 ×100 ug 4 × 70 ul Buffer modified mRNA 3 Lipoplex-human EPO S.C. 4 × 100ug + 30% Lipoplex 4 × 70 ul Lipoplex modified mRNA 4 Lipoplex-human EPOS.C. 4 × 100 ug 4 × 70 ul Buffer modified mRNA 5 Lipoplex-human EPO I.V.200 ug + 30% Lipoplex 140 ul Lipoplex modified mRNA 6Lipoplexed-Luciferase I.M. 100 ug + 30% Lipoplex 4 × 70 ul Lipoplexmodified mRNA 7 Lipoplexed-Luciferase I.M. 100 ug 4 × 70 ul Buffermodified mRNA 8 Lipoplexed-Luciferase S.C. 100 ug + 30% Lipoplex 4 × 70ul Lipoplex modified mRNA 9 Lipoplexed-Luciferase S.C. 100 ug 4 × 70 ulBuffer modified mRNA 10 Lipoplexed-human EPO I.V. 200 ug + 30% Lipoplex140 ul Lipoplex modified mRNA 11 Formulation Buffer I.M. 4x multi dosing4 × 70 ul Buffer

TABLE 23 Human EPO Protein in Mouse Serum (I.M. Injection Route) EPO(pg/ml) Formulation I.M. Injection Route S.C. Injection RouteEpo-Lipoplex 67.1 2.2 Luc-Lipoplex 0 0 Epo-Saline 100.9 11.4 Luc-Saline0 0 Formulation Buffer 0 0

Example 76 In Vivo Delivery Using Varying Lipid Ratios

Modified mRNA was delivered to C57/BL6 mice to evaluate varying lipidratios and the resulting protein expression. Formulations of 100 μgmodified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 5; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1;fully modified with 5-methylcytosine and pseudouridine) lipoplexed with10%, 30% or 50% RNAIMAX™, 100 μg modified luciferase mRNA (IVT cDNAsequence shown in SEQ ID NO: 2; mRNA sequence shown in SEQ ID NO: 3,polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap 1, fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site) lipoplexed with 10%, 30%or 50% RNAIMAX™ or a formulation buffer were administeredintramuscularly to mice in a single 70 μl dose. Serum was collected 13hours post injection to undergo a human EPO ELISA to determine the humanEPO protein level in each mouse. The results of the human EPO ELISA,shown in Table 24, show that modified human EPO expressed in the muscleis secreted into the serum for each of the different percentage ofRNAIMAX™.

TABLE 24 Human EPO Protein in Mouse Serum (IM Injection Route)Formulation EPO (pg/ml) Epo + 10% RNAiMAX 11.4 Luc + 10% RNAiMAX 0 Epo +30% RNAiMAX 27.1 Luc + 30% RNAiMAX 0 Epo + 50% RNAiMAX 19.7 Luc + 50%RNAiMAX 0 F. Buffer 0

Example 77 In Vivo Delivery of Modified RNA in Rats

Protein production of modified mRNA was evaluated by delivering modifiedG-CSF mRNA or modified Factor IX mRNA to female Sprague Dawley rats(n=6). Rats were injected with 400 ug in 100 ul of G-CSF mRNA (mRNAsequence shown in SEQ ID NO: 1; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap 1) fully modified with5-methylcytosine and pseudouridine (G-CSF Gen1), G-CSF mRNA fullymodified with 5-methylcytosine and N1-methylpseudouridine (G-CSF Gen2)or Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1)fully modified with 5-methylcytosine and pseudouridine (Factor IX Gen1)reconstituted from the lyophilized form in 5% sucrose. Blood wascollected 8 hours after injection and the G-CSF protein level in serumwas measured by ELISA. Table 25 shows the G-CSF protein levels in serumafter 8 hours.

These results demonstrate that both G-CSF Gen 1 and G-CSF Gen 2 modifiedmRNA can produce human G-CSF protein in a rat following a singleintramuscular injection, and that human G-CSF protein production isimproved when using Gen 2 chemistry over Gen 1 chemistry.

TABLE 25 G-CSF Protein in Rat Serum (I.M. Injection Route) FormulationG-CSF protein (pg/ml) G-CSF Gen1 19.37 G-CSF Gen2 64.72 Factor IX Gen 12.25

Example 78 Chemical Modification: In Vitro Studies A. In Vitro Screeningin PBMC

500 ng of G-CSF (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1) mRNAfully modified with the chemical modification outlined Tables 26 and 27was transfected with 0.4 uL Lipofectamine 2000 into peripheral bloodmononuclear cells (PBMC) from three normal blood donors. Control samplesof LPS, R848, P(I)P(C) and mCherry (mRNA sequence shown in SEQ ID NO: 4;polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap 1; fully modified with 5-methylcytosine and pseudouridine) werealso analyzed. The supernatant was harvested and stored frozen untilanalyzed by ELISA to determine the G-CSF protein expression, and theinduction of the cytokines interferon-alpha (IFN-α) and tumor necrosisfactor alpha (TNF-α). The protein expression of G-CSF is shown in Table26, the expression of IFN-α and TNF-α is shown in Table 27.

The data in Table 26 demonstrates that many, but not all, chemicalmodifications can be used to productively produce human G-CSF in PBMC.Of note, 100% N1-methylpseudouridine substitution demonstrates thehighest level of human G-CSF production (almost 10-fold higher thanpseudouridine itself). When N1-methylpseudouridine is used incombination with 5-methylcytidine a high level of human G-CSF protein isalso produced (this is also higher than when pseudouridine is used incombination with 5 methylcytidine).

Given the inverse relationship between protein production and cytokineproduction in PBMC, a similar trend is also seen in Table 27, where 100%substitution with N1-methylpseudouridine results no cytokine induction(similar to transfection only controls) and pseudouridine showsdetectable cytokine induction which is above background.

Other modifications such as N6-methyladenosine and α-thiocytidine appearto increase cytokine stimulation.

TABLE 26 Chemical Modifications and G-CSF Protein Expression G-CSFProtein Expression (pg/ml) Donor Donor Donor Chemical Modifications 1 23 Pseudouridine 2477 1,909 1,498 5-methyluridine 318 359 345N1-methylpseudouridine 21,495 16,550 12,441 2-thiouridine 932 1,000 6004-thiouridine 5 391 218 5-methoxyuridine 2,964 1,832 1,8005-methylcytosine and pseudouridine 2,632 1,955 1,373 (1^(st) set)5-methylcytosine and N1-methyl- 10,232 7,245 6,214 pseudouridine (1^(st)set) 2′Fluoroguanosine 59 186 177 2′Fluorouridine 118 209 1915-methylcytosine and pseudouridine 1,682 1,382 1,036 (2^(nd) set)5-methylcytosine and N1-methyl- 9,564 8,509 7,141 pseudouridine (2^(nd)set) 5-bromouridine 314 482 291 5-(2-carbomethoxyvinyl)uridine 77 286177 5-[3(1-E-propenylamino)uridine 541 491 550 α-thiocytidine 105 264245 5-methylcytosine and pseudouridine 1,595 1,432 955 (3^(rd) set)N1-methyladenosine 182 177 191 N6-methyladenosine 100 168 2005-methylcytidine 291 277 359 N4-acetylcytidine 50 136 365-formylcytidine 18 205 23 5-methylcytosine and pseudouridine 264 350182 (4^(th) set) 5-methylcytosine and N1-methyl- 9,505 6,927 5,405pseudouridine (4^(th) set) LPS 1,209 786 636 mCherry 5 168 164 R848 709732 636 P(I)P(C) 5 186 182

TABLE 27 Chemical Modifications and Cytokine Expression IFN-α Expression(pg/ml) TNF-α Expression (pg/ml) Chemical Modifications Donor 1 Donor 2Donor 3 Donor 1 Donor 2 Donor 3 Pseudouridine 120 77 171 36 81 1265-methyluridine 245 135 334 94 100 157 N1-methylpseudouridine 26 75 138101 106 134 2-thiouridine 100 108 154 133 133 141 4-thiouridine 463 258659 169 126 254 5-methoxyuridine 0 64 133 39 74 111 5-methylcytosine and88 94 148 64 89 121 pseudouridine (1^(st) set) 5-methylcytosine and N1-0 60 136 54 79 126 methylpseudouridine (1^(st) set) 2′Fluoroguanosine107 97 194 91 94 141 2′Fluorouridine 158 103 178 164 121 1565-methylcytosine and 133 92 167 99 111 150 pseudouridine (2^(nd) set)5-methylcytosine and N1- 0 66 140 54 97 149 methylpseudouridine (2^(nd)set) 5-bromouridine 95 86 181 87 106 157 5-(2- 0 61 130 40 81 116carbomethoxyvinyl)uridine 5-[3(1-E- 0 58 132 71 90 119propenylamino)uridine α-thiocytidine 1,138 565 695 300 273 2775-methylcytosine and 88 75 150 84 89 130 pseudouridine (3^(rd) set)N1-methyladenosine 322 255 377 256 157 294 N6-methyladenosine 1,9351,065 1,492 1,080 630 857 5-methylcytidine 643 359 529 176 136 193N4-acetylcytidine 789 593 431 263 67 207 5-formylcytidine 180 93 88 13630 40 5-methylcytosine and 131 28 18 53 24 29 pseudouridine (4^(th) set)5-methylcytosine and N1- 0 0 0 36 14 13 methylpseudouridine (4^(th) set)LPS 0 67 146 7,004 3,974 4,020 mCherry 100 75 143 67 100 133 R848 674619 562 11,179 8,546 9,907 P(I)P(C) 470 117 362 249 177 197

B. In vitro Screening in HeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37oG in 5% CO₂ atmosphere overnight. Next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown in SEQ ID NO: 3; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1)with the chemical modification described in Table 28, were diluted in 10ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul were diluted in 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 28. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

These results demonstrate that many, but not all, chemical modificationscan be used to productively produce human G-CSF in HeLa cells. Of note,100% N1-methylpseudouridine substitution demonstrates the highest levelof human G-CSF production.

TABLE 28 Relative Light Units of Luciferase Chemical Modification RLUN6-methyladenosine (m6a) 534 5-methylcytidine (m5c) 138,428N4-acetylcytidine (ac4c) 235,412 5-formylcytidine (f5c) 4365-methylcytosine/pseudouridine, test A1 48,6595-methylcytosine/N1-methylpseudouridine, test A1 190,924 Pseudouridine655,632 1-methylpseudouridine (m1u) 1,517,998 2-thiouridine (s2u) 33875-methoxyuridine (mo5u) 253,719 5-methylcytosine/pseudouridine, test B1317,744 5-methylcytosine/N1-methylpseudouridine, test B1 265,8715-Bromo-uridine 43,276 5 (2 carbovinyl) uridine 531 5 (3-1E propenylAmino) uridine 446 5-methylcytosine/pseudouridine, test A2 295,8245-methylcytosine/N1-methylpseudouridine, test A2 233,921 5-methyluridine50,932 α-Thio-cytidine 26,358 5-methylcytosine/pseudouridine, test B2481,477 5-methylcytosine/N1-methylpseudouridine, test B2 271,9895-methylcytosine/pseudouridine, test A3 438,8315-methylcytosine/N1-methylpseudouridine, test A3 277,499 UnmodifiedLuciferase 234,802

C. In Vitro Screening in Rabbit Reticulocyte Lysates

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1) wasmodified with the chemical modification listed in Table 29 and werediluted in sterile nuclease-free water to a final amount of 250 ng in 10ul. The diluted luciferase was added to 40 ul of freshly prepared RabbitReticulocyte Lysate and the in vitro translation reaction was done in astandard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific,Waltham, Mass.) at 30° C. in a dry heating block. The translation assaywas done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit(Promega, Madison, Wis.) according to the manufacturer's instructions.The reaction buffer was supplemented with a one-to-one blend of providedamino acid stock solutions devoid of either Leucine or Methionineresulting in a reaction mix containing sufficient amounts of both aminoacids to allow effective in vitro translation.

After 60 minutes of incubation, the reaction was stopped by placing thereaction tubes on ice. Aliquots of the in vitro translation reactioncontaining luciferase modified RNA were transferred to white opaquepolystyrene 96-well plates (Corning, Manassas, Va.) and combined with100 ul complete luciferase assay solution (Promega, Madison, Wis.). Thevolumes of the in vitro translation reactions were adjusted or diluteduntil no more than 2 mio relative light units (RLUs) per well weredetected for the strongest signal producing samples and the RLUs foreach chemistry tested are shown in Table 29. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

These cell-free translation results very nicely correlate with theprotein production results in HeLa, with the same modificationsgenerally working or not working in both systems. One notable exceptionis 5-formylcytidine modified luciferase mRNA which worked in thecell-free translation system, but not in the HeLa cell-basedtransfection system. A similar difference between the two assays wasalso seen with 5-formylcytidine modified G-CSF mRNA.

TABLE 29 Relative Light Units of Luciferase Chemical Modification RLUN6-methyladenosine (m6a) 398 5-methylcytidine (m5c) 152,989N4-acetylcytidine (ac4c) 60,879 5-formylcytidine (f5c) 55,2085-methylcytosine/pseudouridine, test A1 349,3985-methylcytosine/N1-methylpseudouridine, test A1 205,465 Pseudouridine587,795 1-methylpseudouridine (m1u) 589,758 2-thiouridine (s2u) 7085-methoxyuridine (mo5u) 288,647 5-methylcytosine/pseudouridine, test B1454,662 5-methylcytosine/N1-methylpseudouridine, test B1 223,7325-Bromo-uridine 221,879 5 (2 carbovinyl) uridine 225 5 (3-1E propenylAmino) uridine 211 5-methylcytosine/pseudouridine, test A2 558,7795-methylcytosine/N1-methylpseudouridine, test A2 333,082 5-methyluridine214,680 α-Thio-cytidine 123,878 5-methylcytosine/pseudouridine, test B2487,805 5-methylcytosine/N1-methylpseudouridine, test B2 154,0965-methylcytosine/pseudouridine, test A3 413,5355-methylcytosine/N1-methylpseudouridine, test A3 292,954 UnmodifiedLuciferase 225,986

Example 79 Chemical Modification: In Vivo Studies A. In Vivo Screeningof G-CSF Modified mRNA

Balb-C mice (n=4) are intramuscularly injected in each leg with modifiedG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),fully modified with the chemical modifications outlined in Table 30, isformulated in 1×PBS. A control of luciferase modified mRNA (mRNAsequence shown in SEQ ID NO: 3; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified withpseudouridine and 5-methylcytosine) and a control of PBS are alsotested. After 8 hours serum is collected to determine G-CSF proteinlevels cytokine levels by ELISA.

TABLE 30 G-CSF mRNA Chemical Modifications G-CSF Pseudouridine G-CSF5-methyluridine G-CSF 2-thiouridine G-CSF 4-thiouridine G-CSF5-methoxyuridine G-CSF 2′-fluorouridine G-CSF 5-bromouridine G-CSF5-[3(1-E-propenylamino)uridine) G-CSF alpha-thio-cytidine G-CSF5-methylcytidine G-CSF N4-acetylcytidine G-CSF Pseudouridine and5-methylcytosine G-CSF N1-methylpseudouridine and 5-methylcytosineLuciferase Pseudouridine and 5-methylcytosine PBS None

B. In Vivo Screening of Luciferase Modified mRNA

Balb-C mice (n=4) were subcutaneously injected with 200 ul containing 42to 103 ug of modified luciferase mRNA (mRNA sequence shown in SEQ ID NO:3; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1), fully modified with the chemical modifications outlined inTable 31, was formulated in 1×PBS. A control of PBS was also tested. Thedosages of the modified luciferase mRNA is also outlined in Table 31. 8hours after dosing the mice were imaged to determine luciferaseexpression. Twenty minutes prior to imaging, mice were injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals werethen anesthetized and images were acquired with an IVIS Lumina IIimaging system (Perkin Elmer). Bioluminescence was measured as totalflux (photons/second) of the entire mouse.

As demonstrated in Table 31, all luciferase mRNA modified chemistriesdemonstrated in vivo activity, with the exception of 2′-fluorouridine.In addition 1-methylpseudouridine modified mRNA demonstrated very highexpression of luciferase (5-fold greater expression than pseudouridinecontaining mRNA).

TABLE 31 Luciferase Screening Dose Dose Luciferase Chemical (ug) ofvolume expression mRNA Modifications mRNA (ml) (photon/second)Luciferase 5-methylcytidine 83 0.72 1.94E+07 LuciferaseN4-acetylcytidine 76 0.72 1.11E07  Luciferase Pseudouridine 95 1.201.36E+07 Luciferase 1-methylpseudouridine 103 0.72 7.40E+07 Luciferase5-methoxyuridine 95 1.22 3.32+07   Luciferase 5-methyluridine 94 0.867.42E+06 Luciferase 5-bromouridine 89 1.49 3.75E+07 Luciferase2′-fluoroguanosine 42 0.72 5.88E+05 Luciferase 2′-fluorocytidine 47 0.724.21E+05 Luciferase 2′-flurorouridine 59 0.72 3.47E+05 PBS None — 0.723.16E+05

Example 80 In Vivo Screening of Combination Luciferase Modified mRNA

Balb-C mice (n=4) were subcutaneously injected with 200 ul of 100 ug ofmodified luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1), fully modified with the chemical modifications outlined in Table 32,was formulated in 1×PBS. A control of PBS was also tested. The dosagesof the modified luciferase mRNA is also outlined in Table 29. 8 hoursafter dosing the mice were imaged to determine luciferase expression.Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

As demonstrated in Table 32, all luciferase mRNA modified chemistries(in combination) demonstrated in vivo activity. In addition the presenceof N1-methylpseudouridine in the modified mRNA (with N4-acetylcytidineor 5 methylcytidine) demonstrated higher expression than when the samecombinations where tested using with pseudouridine. Taken together,these data demonstrate that N1-methylpseudouridine containing luciferasemRNA results in improved protein expression in vivo whether used alone(Table 31) or when used in combination with other modified nulceotides(Table 32).

TABLE 32 Luciferase Screening Combinations Luciferase expression mRNAChemical Modifications (photon/second) LuciferaseN4-acetylcytidine/pseudouridine 4.18E+06 LuciferaseN4-acetylcytidine/N1-methyl- 2.88E+07 pseudouridine Luciferase5-methylcytidine/5-methoxyuridine 3.48E+07 Luciferase5-methylcytidine/5-methyluridine 1.44E+07 Luciferase5-methylcytidine/where 50% of the 2.39E+06 uridine is replaced with2-thiouridine Luciferase 5-methylcytidine/pseudouridine 2.36E+07Luciferase 5-methylcytidine/N1-methyl- 4.15E+07 pseudouridine PBS None3.59E+05

Example 81 Stability of Modified RNA A. Storage of Modified RNA

Stability experiments were conducted to obtain a better understanding ofstorage conditions to retain the integrity of modified RNA. UnmodifiedG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine andG-CSF mRNA fully modified with 5-methylcytosine and pseudouridinelipoplexed with 0.75% by volume of RNAIMAX™ was stored at 50° C., 40°C., 37° C., 25° C., 4° C. or −20° C. After the mRNA had been stored for0 hours, 2 hours, 6 hours, 24 hours, 48 hours, 5 days and 14 days, themRNA was analyzed by gel electrophoresis using a Bio-Rad EXPERION™system. The modified, unmodified and lipoplexed G-CSF mRNA was alsostored in RNASTABLE® (Biomatrica, Inc. San Diego, Calif.) at 40° C. orwater at −80° C. or 40° C. for 35 days before being analyzed by gelelectrophoresis.

All mRNA samples without stabilizer were stable after 2 weeks afterstorage at 4° C. or −20° C. Modified G-CSF mRNA, with or withoutlipoplex, was more stable than unmodified G-CSF when stored at 25° C.(stable out to 5 days versus 48 hours), 37° C. (stable out to 24 hoursversus 6 hours) and 50° C. (stable out to 6 hours versus 2 hours).Unmodified G-CSF mRNA, modified G-CSF mRNA with or without lipoplextolerated 12 freeze/thaw cycles.

mRNA samples stored in stabilizer at 40° C. showed similar stability tothe mRNA samples stored in water at −80° C. after 35 days whereas themRNA stored in water at 40° C. showed heavy degradation after 18 days.

Example 82 Cell Viability in BJ Fibroblasts

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, cat#30-2003) supplemented with10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts wereseeded on a 24-well plate at a density of 130,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap 1) fully modified with 5-methylcytosine andpseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methylpseudouridine (Gen2) was transfected using Lipofectamine 2000(Invitrogen, cat#11668-019), following manufacturer's protocol. Controlsamples of Lipofectamine 2000 (LF2000) and unmodified G-CSF mRNA werealso transfected. The modified mRNA or control samples were transfecteddaily for 4 days. The viability of the cells after transfection wasevaluated 6 hours and 24 hours after the first transfection (Ti, 6 hoursor T1, 24 hours), and 24 hours after the second (T2, 24 hours) andfourth transfection (T4, 24 hours).

To determine cell viability, the culture medium was completely removedand the cells were washed once with 600 ul of sterile PBS withoutCa2+/Mg2+ (Gibco/Life Technologies, Manassas, Va.) in order to rinse-offloosely attached cells. PBS was removed and discarded. The cleanedfibroblasts in each well were treated with 220 ul of a diluted CELLTITER GLO® (Promega, catalog #G7570) stock solution (the CELL TITER GLO®stock solution was further diluted 1:1 with an equal amount of sterilePBS). A sterile pipet tip was used to scratch the cells off the plateand accelerate the lysis process.

For two time intervals, T1, 24 hours and T2, 24 hours, an alternativeprotocol was applied. Cells were washed with PBS, as described above,and subsequently trypsinized with Trypsin/EDTA solution (Gibco/LifeTechnologies, Manassas, Va.). Cells were detached and collected in 500ul of medium containing trypsin inhibitor. Cells were harvested bycentrifugation at 1200 rcf for 5 minutes. The cell pellet wasresuspended in 500 ul PBS. This cell suspension was kept on ice, and 100ul of this was combined with 100 ul of undiluted Cell Titer Glosolution.

All of the CELL TITER GLO® lysates were then incubated at roomtemperature for 20 minutes. 20 ul of the lysates were transferred to awhite opaque polystyrene 96-well plate (Corning, Manassas, Va.) andcombined with 100 ul diluted CELL TITER GLO® solution. The plate readerused was from BioTek Synergy H1 (BioTek, Winooski, Vt.) and the absolutevalues were normalized to signal of the untreated BJ Fibroblasts to 100%cell vitality. The percent viability for the BJ fibroblasts are shown inTable 33.

Importantly, all of these experiments are conducted in the absence ofany interferon or other cytokine inhibitors and thus represent anaccurate measure of the cytotoxicity of the different mRNA.

These results demonstrate that repeated transfection of BJ fibroblastswith unmodified mRNA results in loss of cell viability that is apparentas early as 24 hrs after the first transfection (Ti, 24 hours) andcontinues to be apparent and more pronounced at subsequent time points.

There is also a loss of viability with repeated transfection of5-methylcytidine and pseudouridine modified mRNA that is apparent 24hours after the fourth daily transfection (T4, 24 hours). No loss ofcell viability over the course of this experiment is seen using5-methylcytidine and N1-methylpseudouridine modified mRNA. These resultsdemonstrate that 5-methylcytidine and N1-methylpseudouridine containingmRNA have improved cell viability when analyzed under repeatedtransfection. The ability to repeatedly administer modified mRNA isimportant in most therapeutic applications, and as such the ability todo so without cytotoxicity is also important. While not wishing to bebound by theory, it is believed that response genes following a singletransfection may lead to a decrease in protein production, cytokineinduction, and eventually loss of cell viability. These results areconsistent with N1-methylpseudouridine-containing mRNA showing animproved profile in this respect relative to both unmodified mRNA andpseudouridine-modified mRNA.

TABLE 33 Percent Viability T1, 6 hours T1, 24 hours T2, 24 hours T4, 24hours Gen 1 G-CSF 81 108 91 65 Gen 2 G-CSF 99 102 128 87 UnmodifiedG-CSF 101 72 74 42 LF2000 99 80 114 106 Untreated 100 100 100 100

Example 83 Innate Immune Response in BJ Fibroblasts

Human primary foreskin fibroblasts (BJ fibroblasts) are obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, cat#30-2003) supplemented with10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts areseeded on a 24-well plate at a density of 130,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 1; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methylpseudouridine (Gen2) is transfected using Lipofectamine 2000(Invitrogen, cat#11668-019), following manufacturer's protocol. Controlsamples of Lipofectamine 2000 and unmodified G-CSF mRNA (natural G-CSF)are also transfected. The cells are transfected for five consecutivedays. The transfection complexes are removed four hours after each roundof transfection.

The culture supernatant is assayed for secreted GCSF (R&D Systems,catalog #DCS50), tumor necrosis factor-alpha (TNF-alpha) and interferonalpha (IFN-alpha) by ELISA every day after transfection followingmanufacturer's protocols. The cells are analyzed for viability usingCELL TITER GLO® (Promega, catalog #G7570) 6 hrs and 18 hrs after thefirst round of transfection and every alternate day following that. Atthe same time from the harvested cells, total RNA is isolated andtreated with DNASE® using the RNAEASY micro kit (catalog #74004)following the manufacturer's protocol. 100 ng of total RNA is used forcDNA synthesis using the High Capacity cDNA Reverse Transcription kit(Applied Biosystems, cat #4368814) following the manufacturer'sprotocol. The cDNA is then analyzed for the expression of innate immuneresponse genes by quantitative real time PCR using SybrGreen in a BioradCFX 384 instrument following the manufacturer's protocol.

Example 84 In Vitro Transcription with Wild-Type T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1) werefully modified with different chemistries and chemistry combinationslisted in Tables 34-37 using wild-type T7 polymerase as previouslydescribed.

The yield of the translation reactions was determined byspectrophometric measurement (OD260) and the yield for Luciferase isshown in Table 34 and G-CSF is shown in Table 36.

The luciferase and G-CSF modified mRNA were also subjected to anenzymatic capping reaction and each modified mRNA capping reaction wasevaluated for yield by spectrophometic measurement (OD260) and correctsize assessed using bioanalyzer. The yield from the capping reaction forluciferase is shown in Table 35 and G-CSF is shown in Table 37.

TABLE 34 In vitro transcription chemistry for Luciferase Yield ChemicalModification (mg) N6-methyladenosine 0.99 5-methylcytidine 1.29N4-acetylcytidine 1.0 5-formylcytidine 0.55 Pseudouridine 2.0N1-methylpseudouridine 1.43 2-thiouridine 1.56 5-methoxyuridine 2.355-methyluridine 1.01 α-Thio-cytidine 0.83 5-Br-uridine (5Bru) 1.96 5 (2carbomethoxyvinyl) uridine 0.89 5 (3-1E propenyl Amino) uridine 2.01N4-acetylcytidine/pseudouridine 1.34N4-acetylcytidine/N1-methylpseudouridine 1.265-methylcytidine/5-methoxyuridine 1.38 5-methylcytidine/5-bromouridine0.12 5-methylcytidine/5-methyluridine 2.97 5-methylcytidine/half of theuridines are modified 1.59 with 2-thiouridine5-methylcytidine/2-thiouridine 0.90 5-methylcytidine/pseudouridine 1.835-methylcytidine/N1 methyl pseudouridine 1.33

TABLE 35 Capping chemistry and yield for Luciferase modified mRNA YieldChemical Modification (mg) 5-methylcytidine 1.02 N4-acetylcytidine 0.935-formylcytidine 0.55 Pseudouridine 2.07 N1-methylpseudouridine 1.272-thiouridine 1.44 5-methoxyuridine 2 5-methyluridine 0.8α-Thio-cytidine 0.74 5-Br-uridine (5Bru) 1.29 5 (2 carbomethoxyvinyl)uridine 0.54 5 (3-1E propenyl Amino) uridine 1.39N4-acetylcytidine/pseudouridine 0.99N4-acetylcytidine/N1-methylpseudouridine 1.085-methylcytidine/5-methoxyuridine 1.13 5-methylcytidine/5-methyluridine1.08 5-methylcytidine/half of the uridines are modified 1.2 with2-thiouridine 5-methylcytidine/2-thiouridine 1.275-methylcytidine/pseudouridine 1.19 5-methylcytidine/N1 methylpseudouridine 1.04

TABLE 36 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Yield Chemical Modification (mg) N6-methyladenosine 1.575-methylcytidine 2.05 N4-acetylcytidine 3.13 5-formylcytidine 1.41Pseudouridine 4.1 N1-methylpseudouridine 3.24 2-thiouridine 3.465-methoxyuridine 2.57 5-methyluridine 4.27 4-thiouridine 1.452′-F-uridine 0.96 α-Thio-cytidine 2.29 2′-F-guanosine 0.6N-1-methyladenosine 0.63 5-Br-uridine (5Bru) 1.08 5 (2carbomethoxyvinyl) uridine 1.8 5 (3-1E propenyl Amino) uridine 2.09N4-acetylcytidine/pseudouridine 1.72N4-acetylcytidine/N1-methylpseudouridine 1.375-methylcytidine/5-methoxyuridine 1.85 5-methylcytidine/5-methyluridine1.56 5-methylcytidine/half of the uridines are modified 1.84 with2-thiouridine 5-methylcytidine/2-thiouridine 2.535-methylcytidine/pseudouridine 0.63 N4-acetylcytidine/2-thiouridine 1.3N4-acetylcytidine/5-bromouridine 1.37 5-methylcytidine/N1 methylpseudouridine 1.25 N4-acetylcytidine/pseudouridine 2.24

TABLE 37 Capping chemistry and yield for G-CSF modified mRNA ChemicalModification Yield (mg) N6-methyladenosine 1.04 5-methylcytidine 1.08N4-acetylcytidine 2.73 5-formylcytidine 0.95 Pseudouridine 3.88N1-methylpseudouridine 2.58 2-thiouridine 2.57 5-methoxyuridine 2.055-methyluridine 3.56 4-thiouridine 0.91 2′-F-uridine 0.54α-Thio-cytidine 1.79 2′-F-guanosine 0.14 5-Br-uridine (5Bru) 0.79 5 (2carbomethoxyvinyl) uridine 1.28 5 (3-1E propenyl Amino) uridine 1.78N4-acetylcytidine/pseudouridine 0.29N4-acetylcytidine/N1-methylpseudouridine 0.335-methylcytidine/5-methoxyuridine 0.91 5-methylcytidine/5-methyluridine0.61 5-methylcytidine/half of the uridines are modified 1.24 with2-thiouridine 5-methylcytidine/pseudouridine 1.08N4-acetylcytidine/2-thiouridine 1.34 N4-acetylcytidine/5-bromouridine1.22 5-methylcytidine/N1 methyl pseudouridine 1.56

Example 85 In Vitro Transcription with Mutant T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1) werefully modified with different chemistries and chemistry combinationslisted in Tables 38-41 using a mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).

The yield of the translation reactions was determined byspectrophometric measurement (OD260) and the yield for Luciferase isshown in Table 38 and G-CSF is shown in Table 40.

The luciferase and G-CSF modified mRNA were also subjected to anenzymatic capping reaction and each modified mRNA capping reaction wasevaluated for yield by spectrophometic measurement (OD260) and correctsize assessed using bioanalyzer. The yield from the capping reaction forluciferase is shown in Table 39 and G-CSF is shown in Table 41.

TABLE 38 In vitro transcription chemistry and yield for Luciferasemodified mRNA Chemical Modification Yield (ug) 2′Fluorocytosine 71.42′Fluorouridine 57.5 5-methylcytosine/pseudouridine, test A 26.45-methylcytosine/N1-methylpseudouridine, test A 73.3N1-acetylcytidine/2-fluorouridine 202.2 5-methylcytidine/2-fluorouridine 131.9  2-fluorocytosine/pseudouridine119.3  2-fluorocytosine/N1-methylpseudouridine 107.0 2-fluorocytosine/2-thiouridine 34.7 2-fluorocytosine/5-bromouridine 81.02-fluorocytosine/2-fluorouridine 80.4 2-fluoroguanine/5-methylcytosine61.2 2-fluoroguanine/5-methylcytosine/pseudouridine 65.02-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 41.22-fluoroguanine/pseudouridine 79.12-fluoroguanine/N1-methylpseudouridine 74.65-methylcytidine/pseudouridine, test B 91.85-methylcytidine/N1-methylpseudouridine, test B 72.4 2′fluoroadenosine190.98 

TABLE 39 Capping chemistry and yield for Luciferase modified mRNAChemical Modification Yield (ug) 2′Fluorocytosine 19.2 2′Fluorouridine16.7 5-methylcytosine/pseudouridine, test A 7.05-methylcytosine/N1-methylpseudouridine, test A 21.5N1-acetylcytidine/2-fluorouridine 47.5 5-methylcytidine/2-fluorouridine53.2 2-fluorocytosine/pseudouridine 58.4 2-fluorocytosine/N1-methylpseudouridine 26.22-fluorocytosine/2-thiouridine 12.9 2-fluorocytosine/5-bromouridine 26.52-fluorocytosine/2-fluorouridine 35.7 2-fluoroguanine/5-methylcytosine24.7 2-fluoroguanine/5-methylcytosine/pseudouridine 32.32-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 31.32-fluoroguanine/pseudouridine 20.9 2-fluoroguanine/N1-methylpseudouridine 29.85-methylcytidine/pseudouridine, test B 58.25-methylcytidine/N1-methylpseudouridine, test B 44.4

TABLE 40 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Chemical Modification Yield (ug) 2′Fluorocytosine 56.52′Fluorouridine 79.4 5-methylcytosine/pseudouridine, test A 21.25-methylcytosine/N1-methylpseudouridine, test A 77.1N1-acetylcytidine/2-fluorouridine 168.6 5-methylcytidine/2-fluorouridine134.7 2-fluorocytosine/pseudouridine 97.82-fluorocytosine/N1-methylpseudouridine 103.12-fluorocytosine/2-thiouridine 58.8 2-fluorocytosine/5-bromouridine 88.82-fluorocytosine/2-fluorouridine 93.9 2-fluoroguanine/5-methylcytosine97.3 2-fluoroguanine/5-methylcytosine/pseudouridine 96.02-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 82.02-fluoroguanine/pseudouridine 68.02-fluoroguanine/N1-methylpseudouridine 59.35-methylcytidine/pseudouridine, test B 58.75-methylcytidine/N1-methylpseudouridine, test B 78.0

TABLE 41 Capping chemistry and yield for G-CSF modified mRNA ChemicalModification Yield (ug) 2′Fluorocytosine 16.9 2′Fluorouridine 17.05-methylcytosine/pseudouridine, test A 10.65-methylcytosine/N1-methylpseudouridine, test A 22.7N1-acetylcytidine/2-fluorouridine 19.9 5-methylcytidine/2-fluorouridine21.3 2-fluorocytosine/pseudouridine 65.22-fluorocytosine/N1-methylpseudouridine 58.92-fluorocytosine/2-thiouridine 41.2 2-fluorocytosine/5-bromouridine 35.82-fluorocytosine/2-fluorouridine 36.7 2-fluoroguanine/5-methylcytosine36.6 2-fluoroguanine/5-methylcytosine/pseudouridine 37.32-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 30.72-fluoroguanine/pseudouridine 29.02-fluoroguanine/N1-methylpseudouridine 22.75-methylcytidine/pseudouridine, test B 60.45-methylcytidine/N1-methylpseudouridine, test B 33.0

Example 86 2′O-methyl and 2′Fluoro Compounds

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1) wereproduced as fully modified versions with the chemistries in Table 42 andtranscribed using mutant T7 polymerase (Durascribe® T7 Transcription kit(Cat. No. DS010925) (Epicentre®, Madison, Wis.). 2′ fluoro-containingmRNA were made using Durascribe T7, however, 2′Omethyl-containing mRNAcould not be transcribed using Durascribe T7.

Incorporation of 2′Omethyl modified mRNA might possibly be accomplishedusing other mutant T7 polymerases (Nat. Biotechnol. (2004) 22:1155-1160;Nucleic Acids Res. (2002) 30:e138). Alternatively, 2′OMe modificationscould be introduced post-transcriptionally using enzymatic means.

Introduction of modifications on the 2′ group of the sugar has manypotential advantages. 2′OMe substitutions, like 2′ fluoro substitutionsare known to protect against nucleases and also have been shown toabolish innate immune recognition when incorporated into other nucleicacids such as siRNA and anti-sense (incorporated in its entirety,Crooke, ed. Antisense Drug Technology, 2^(nd) edition; Boca Raton: CRCpress).

The 2′Fluoro-modified mRNA were then transfected into HeLa cells toassess protein production in a cell context and the same mRNA were alsoassessed in a cell-free rabbit reticulocyte system. A control ofunmodified luciferase (natural luciferase) was used for bothtranscription experiments, a control of untreated and mock transfected(Lipofectamine 2000 alone) were also analyzed for the HeLa transfectionand a control of no RNA was analyzed for the rabbit reticulysates.

For the HeLa transfection experiments, the day before transfection,20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested bytreatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island,N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplementedwith 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate(Corning, Manassas, Va.). The cells were grown at 37oG in 5% CO₂atmosphere overnight. Next day, 83 ng of the 2′fluoro-containingluciferase modified RNA (mRNA sequence shown in SEQ ID NO: 3; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1)with the chemical modification described in Table 42, were diluted in 10ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul were diluted in 10 ul final volume ofOPTI-MEM. After minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. After 18 to 22 hours of incubation cells expressingluciferase were lysed with 100 ul of Passive Lysis Buffer (Promega,Madison, Wis.) according to manufacturer instructions. Aliquots of thelysates were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The lysate volumes wereadjusted or diluted until no more than 2 mio relative light units (RLU)per well were detected for the strongest signal producing samples andthe RLUs for each chemistry tested are shown in Table 42. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The backgroundsignal of the plates without reagent was about 200 relative light unitsper well.

For the rabbit reticulocyte lysate assay, 2′-fluoro-containingluciferase mRNA were diluted in sterile nuclease-free water to a finalamount of 250 ng in 10 ul and added to 40 ul of freshly prepared RabbitReticulocyte Lysate and the in vitro translation reaction was done in astandard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific,Waltham, Mass.) at 30° C. in a dry heating block. The translation assaywas done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit(Promega, Madison, Wis.) according to the manufacturer's instructions.The reaction buffer was supplemented with a one-to-one blend of providedamino acid stock solutions devoid of either Leucine or Methionineresulting in a reaction mix containing sufficient amounts of both aminoacids to allow effective in vitro translation. After 60 minutes ofincubation, the reaction was stopped by placing the reaction tubes onice.

Aliquots of the in vitro translation reaction containing luciferasemodified RNA were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The volumes of the in vitrotranslation reactions were adjusted or diluted until no more than 2 miorelative light units (RLUs) per well were detected for the strongestsignal producing samples and the RLUs for each chemistry tested areshown in Table 43. The plate reader was a BioTek Synergy H1 (BioTek,Winooski, Vt.). The background signal of the plates without reagent wasabout 160 relative light units per well.

As can be seen in Table 42 and 43, multiple 2′Fluoro-containingcompounds are active in vitro and produce luciferase protein.

TABLE 42 HeLa Cells Chemical Concentration Modification (ug/ml) Volume(ul) Yield (ug) RLU 2′Fluoroadenosine 381.96 500 190.98 388.52′Fluorocytosine 654.56 500 327.28 2420 2′Fluoroguanine  541,795 500270.90 11,705.5 2′Flurorouridine 944.005 500 472.00 6767.5 Naturalluciferase N/A N/A N/A 133,853.5 Mock N/A N/A N/A 340 Untreated N/A N/AN/A 238

TABLE 43 Rabbit Reticulysates Chemical Modification RLU2′Fluoroadenosine 162 2′Fluorocytosine 208 2′Fluoroguanine 371,5092′Flurorouridine 258 Natural luciferase 2,159,968 No RNA 156

Example 87 Luciferase in HeLa Cells Using a Combination of Modifications

To evaluate using of 2′fluoro-modified mRNA in combination with othermodification a series of mRNA were transcribed using either wild-type T7polymerase (non-fluoro-containing compounds) or using mutant T7polymerases (fluyoro-containing compounds) as described in Example 86.All modified mRNA were tested by in vitro transfection in HeLa cells.

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37oG in 5% CO₂ atmosphere overnight. Next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown in SEQ ID NO: 3; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1)with the chemical modification described in Table 44, were diluted in 10ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul were diluted in 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 44. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

As evidenced in Table 44, most combinations of modifications resulted inmRNA which produced functional luciferase protein, including all thenon-fluoro containing compounds and many of the combinations containing2′fluoro modifications.

TABLE 44 Luciferase Chemical Modification RLUN4-acetylcytidine/pseudouridine 113,796N4-acetylcytidine/N1-methylpseudouridine 316,3265-methylcytidine/5-methoxyuridine 24,9485-methylcytidine/5-methyluridine 43,675 5-methylcytidine/half of theuridines modified with 41,601 50% 2-thiouridine5-methylcytidine/2-thiouridine 1,102 5-methylcytidine/pseudouridine51,035 5-methylcytidine/N1 methyl pseudouridine 152,151N4-acetylcytidine/2′Fluorouridine triphosphate 2885-methylcytidine/2′Fluorouridine triphosphate 269 2′Fluorocytosinetriphosphate/pseudouridine 260 2′Fluorocytosinetriphosphate/N1-methylpseudouridine 412 2′Fluorocytosinetriphosphate/2-thiouridine 427 2′Fluorocytosinetriphosphate/5-bromouridine 253 2′Fluorocytosinetriphosphate/2′Fluorouridine triphosphate 184 2′Fluoroguaninetriphosphate/5-methylcytidine 321 2′Fluoroguaninetriphosphate/5-methylcytidine/Pseudouridine 2072′Fluoroguanine/5-methylcytidine/N1 methylpsuedouridine 2352′Fluoroguanine/pseudouridine 218 2′Fluoroguanine/N1-methylpsuedouridine247 5-methylcytidine/pseudouridine, test A 13,8335-methylcytidine/N-methylpseudouridine, test A 598 2′Fluorocytosinetriphosphate 201 2′Fluorouridine triphosphate 3055-methylcytidine/pseudouridine, test B 115,4015-methylcytidine/N-methylpseudouridine, test B 21,034 Natural luciferase30,801 Untreated 344 Mock 262

Example 88 G-CSF In Vitro Transcription

To assess the activity of all our different chemical modifications inthe context of a second open reading frame, we replicated experimentspreviously conducted using luciferase mRNA, with human G-CSF mRNA. G-CSFmRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap 1) were fullymodified with the chemistries in Tables 45 and 46 using wild-type T7polymerase (for all non-fluoro-containing compounds) or mutant T7polymerase (for all fluoro-containing compounds). The mutant T7polymerase was obtained commercially (Durascribe® T7 Transcription kit(Cat. No. DS010925) (Epicentre®, Madison, Wis.).

The modified RNA in Tables 45 and 46 were transfected in vitro in HeLacells or added to rabbit reticulysates (250 ng of modified mRNA) asindicated. A control of untreated, mock transfected (transfectionreagent alone), G-CSF fully modified with 5-methylcytosine andN1-methylpseudouridine or luciferase control (mRNA sequence shown in SEQID NO: 3; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap 1) fully modified with 5-methylcytosine andN1-methylpseudouridine were also analyzed. The expression of G-CSFprotein was determined by ELISA and the values are shown in Tables 45and 46. In Table 45, “NT” means not tested.

As shown in Table 45, many, but not all, chemical modifications resultedin human G-CSF protein production. These results from cell-based andcell-free translation systems correlate very nicely with the samemodifications generally working or not working in both systems. Onenotable exception is 5-formylcytidine modified G-CSF mRNA which workedin the cell-free translation system, but not in the HeLa cell-basedtransfection system. A similar difference between the two assays wasalso seen with 5-formylcytidine modified luciferase mRNA.

As demonstrated in Table 46, many, but not all, G-CSF mRNA modifiedchemistries (when used in combination) demonstrated in vivo activity. Inaddition the presence of N1-methylpseudouridine in the modified mRNA(with N4-acetylcytidine or 5 methylcytidine) demonstrated higherexpression than when the same combinations where tested using withpseudouridine. Taken together, these data demonstrate thatN1-methylpseudouridine containing G-CSF mRNA results in improved proteinexpression in vitro.

TABLE 45 G-CSF Expression G-CSF protein G-CSF (pg/ml) protein Rabbit(pg/ml) reticulysates Chemical Modification HeLa cells cellsPseudouridine 1,150,909 147,875 5-methyluridine 347,045 147,2502-thiouridine 417,273 18,375 N1-methylpseudouridine NT 230,0004-thiouridine 107,273 52,375 5-methoxyuridine 1,715,909 201,7505-methylcytosine/pseudouridine, Test A 609,545 119,7505-methylcytosine/N1-methylpseudouridine, 1,534,318 110,500 Test A2′-Fluoro-guanosine 11,818 0 2′-Fluoro-uridine 60,455 05-methylcytosine/pseudouridine, Test B 358,182 57,8755-methylcytosine/N1-methylpseudouridine, 1,568,636 76,750 Test B5-Bromo-uridine 186,591 72,000 5-(2carbomethoxyvinyl) uridine 1,364 05-[3(1-E-propenylamino) uridine 27,955 32,625 α-thio-cytidine 120,45542,625 5-methylcytosine/pseudouridine, Test C 882,500 49,250N1-methyl-adenosine 4,773 0 N6-methyl-adenosine 1,591 05-methyl-cytidine 646,591 79,375 N4-acetylcytidine 39,545 8,0005-formyl-cytidine 0 24,000 5-methylcytosine/pseudouridine, Test D 87,04547,750 5-methylcytosine/N1-methylpseudouridine, 1,168,864 97,125 Test DMock 909 682 Untreated 0 0 5-methylcytosine/N1-methylpseudouridine,1,106,591 NT Control Luciferase control NT 0

TABLE 46 Combination Chemistries in HeLa cells G-CSF protein (pg/ml)Chemical Modification HeLa cells N4-acetylcytidine/pseudouridine 537,273N4-acetylcytidine/N1-methylpseudouridine 1,091,8185-methylcytidine/5-methoxyuridine 516,1365-methylcytidine/5-bromouridine 48,864 5-methylcytidine/5-methyluridine207,500 5-methylcytidine/2-thiouridine 33,409N4-acetylcytidine/5-bromouridine 211,591 N4-acetylcytidine/2-thiouridine46,136 5-methylcytosine/pseudouridine 301,3645-methylcytosine/N1-methylpseudouridine 1,017,727N4-acetylcytidine/2′Fluorouridine triphosphate 62,2735-methylcytidine/2′Fluorouridine triphosphate 49,318 2′Fluorocytosinetriphosphate/pseudouridine 7,955 2′Fluorocytosine triphosphate/N1- 1,364methylpseudouridine 2′Fluorocytosine triphosphate/2-thiouridine 02′Fluorocytosine triphosphate/5-bromouridine 1,818 2′Fluorocytosinetriphosphate/2′Fluorouridine 909 triphosphate 2′Fluoroguaninetriphosphate/5-methylcytidine 0 2′Fluoroguaninetriphosphate/5-methylcytidine/ 0 pseudouridine 2′Fluoroguaninetriphosphat/5-methylcytidine/N1 1,818 methylpseudouridine2′Fluoroguanine triphosphate/pseudouridine 1,136 2′Fluoroguaninetriphosphate/2′Fluorocytosine 0 triphosphate/N1-methylpseudouridine5-methylcytidine/pseudouridine 617,7275-methylcytidine/N1-methylpseudouridine 747,0455-methylcytidine/pseudouridine 475,4555-methylcytidine/N1-methylpseudouridine 689,0915-methylcytosine/N1-methylpseudouridine, Control 1 848,4095-methylcytosine/N1-methylpseudouridine, Control 2 581,818 Mock 682Untreated 0 Luciferase 2′Fluorocytosine triphosphate 0 Luciferase2′Fluorouridine triphosphate 0

Example 89 Screening of Chemistries

The tables listed in below (Tables 47-49) summarize much of the in vitroand in vitro screening data with the different compounds presented inthe previous examples. A good correlation exists between cell-based andcell-free translation assays. The same chemistry substitutions generallyshow good concordance whether tested in the context of luciferase orG-CSF mRNA. Lastly, N1-methylpseudouridine containing mRNA show a veryhigh level of protein expression with little to no detectable cytokinestimulation in vitro and in vivo, and is superior to mRNA containingpseudouridine both in vitro and in vivo.

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 3; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 1; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1) weremodified with naturally and non-naturally occurring chemistriesdescribed in Tables 47 and 48 or combination chemistries described inTable 48 and tested using methods described herein.

In Tables 47 and 48, “*” refers to in vitro transcription reaction usinga mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No.DS010925) (Epicentre®, Madison, Wis.); “**” refers to the second resultin vitro transcription reaction using a mutant T7 polymerase(Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®,Madison, Wis.); “***” refers to production seen in cell freetranslations (rabbit reticulocyte lysates); the protein production ofHeLa is judged by “+,” “+/−” and “−”; when referring to G-CSF PBMC“++++” means greater than 6,000 pg/ml G-CSF, “+++” means greater than3,000 pg/ml G-CSF, “++” means greater than 1,500 pg/ml G-CSF, “+” meansgreater than 300 pg/ml G-CSF, “+/−” means 150-300 pg/ml G-CSF and thebackground was about 110 pg/ml; when referring to cytokine PBMC “++++”means greater than 1,000 pg/ml interferon-alpha (IFN-alpha), “+++” meansgreater than 600 pg/ml IFN-alpha, “++” means greater than 300 pg/mlIFN-alpha, “+” means greater than 100 pg/ml IFN-alpha, “−” means lessthan 100 pg/ml and the background was about 70 pg/ml; and “NT” means nottested. In Table 48, the protein production was evaluated using a mutantT7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925)(Epicentre®, Madison, Wis.).

TABLE 47 Naturally Occurring Protein Protein Protein Cytokines In VivoIn Vivo Common Name IVT IVT (Luc; (G-CSF; (G-CSF; (G-CSF; ProteinProtein (symbol) (Luc) (G-CSF) HeLa) HeLa) PBMC) PBMC) (Luc) (G-CSF)1-methyladenosine Fail Pass NT − +/− ++ NT NT (m¹A) N⁶-methyladenosinePass Pass − − +/− ++++ NT NT (m⁶A) 2′-O- Fail* Not Done NT NT NT NT NTNT methyladenosine (Am) 5-methylcytidine Pass Pass + + + ++ + NT (m⁵C)2′-O-methylcytidine Fail* Not Done NT NT NT NT NT NT (Cm) 2-thiocytidine(s²C) Fail Fail NT NT NT NT NT NT N⁴-acetylcytidine Pass Pass + + +/−+++ + NT (ac⁴C) 5-formylcytidine Pass Pass −*** −*** − + NT NT (f⁵C)2′-O- Fail* Not Done NT NT NT NT NT NT methylguanosine (Gm) inosine (I)Fail Fail NT NT NT NT NT NT pseudouridine (Y) Pass Pass + + ++ + + NT5-methyluridine Pass Pass + + +/− + NT NT (m⁵U) 2′-O-methyluridine Fail*Not Done NT NT NT NT NT NT (Um) 1- Pass Pass + Not Done ++++ − + NTmethylpseudouridine (m¹Y) 2-thiouridine (s²U) Pass Pass − + + + NT NT4-thiouridine (s⁴U) Fail Pass + +/− ++ NT NT 5-methoxyuridine PassPass + + ++ − + NT (mo⁵U) 3-methyluridine Fail Fail NT NT NT NT NT NT(m³U)

TABLE 48 Non-Naturally Occurring Protein Protein Protein Cytokines InVivo In Vivo IVT IVT (Luc; (G-CSF; (G-CSF; (G-CSF; Protein ProteinCommon Name (Luc) (G-CSF) HeLa) HeLa) PBMC) PBMC) (Luc) (G-CSF)2′-F-ara-guanosine Fail Fail NT NT NT NT NT NT 2′-F-ara-adenosine FailFail NT NT NT NT NT NT 2′-F-ara-cytidine Fail Fail NT NT NT NT NT NT2′-F-ara-uridine Fail Fail NT NT NT NT NT NT 2′-F-guanosine Fail/Pass**Pass/Fail** +** +/− − + + NT 2′-F-adenosine Fail/Pass** Fail/Fail** −**NT NT NT NT NT 2′-F-cytidine Fail/Pass** Fail/Pass** +** NT NT NT + NT2′-F-uridine Fail/Pass** Pass/Pass** +** + +/− + − NT2′-OH-ara-guanosine Fail Fail NT NT NT NT NT NT 2′-OH-ara-adenosine NotDone Not Done NT NT NT NT NT NT 2′-OH-ara-cytidine Fail Fail NT NT NT NTNT NT 2′-OH-ara-uridine Fail Fail NT NT NT NT NT NT 5-Br-Uridine PassPass + + + + + 5-(2- Pass Pass − − +/− − carbomethoxyvinyl) Uridine5-[3-(1-E- Pass Pass − + + − Propenylamino) Uridine (aka Chem 5)N6-(19-Amino- Fail Fail NT NT NT NT NT NT pentaoxanonadecyl) A2-Dimethylamino Fail Fail NT NT NT NT NT NT guanosine 6-Aza-cytidineFail Fail NT NT NT NT NT NT a-Thio-cytidine Pass Pass + + +/− +++ NT NTPseudo-isocytidine NT NT NT NT NT NT NT NT 5-Iodo-uridine NT NT NT NT NTNT NT NT a-Thio-uridine NT NT NT NT NT NT NT NT 6-Aza-uridine NT NT NTNT NT NT NT NT Deoxy-thymidine NT NT NT NT NT NT NT NT a-Thio guanosineNT NT NT NT NT NT NT NT 8-Oxo-guanosine NT NT NT NT NT NT NT NTO6-Methyl- NT NT NT NT NT NT NT NT guanosine 7-Deaza-guanosine NT NT NTNT NT NT NT NT 6-Chloro-purine NT NT NT NT NT NT NT NT a-Thio-adenosineNT NT NT NT NT NT NT NT 7-Deaza-adenosine NT NT NT NT NT NT NT NT5-iodo-cytidine NT NT NT NT NT NT NT NT

In Table 49, the protein production of HeLa is judged by “+,” “+/−” and“−”; when referring to G-CSF PBMC “++++” means greater than 6,000 pg/mlG-CSF, “+++” means greater than 3,000 pg/ml G-CSF, “++” means greaterthan 1,500 pg/ml G-CSF, “+” means greater than 300 pg/ml G-CSF, “+/−”means 150-300 pg/ml G-CSF and the background was about 110 pg/ml; whenreferring to cytokine PBMC “++++” means greater than 1,000 pg/mlinterferon-alpha (IFN-alpha), “+++” means greater than 600 pg/mlIFN-alpha, “++” means greater than 300 pg/ml IFN-alpha, “+” meansgreater than 100 pg/ml IFN-alpha, “−” means less than 100 pg/ml and thebackground was about 70 pg/ml; “WT” refers to the wild type T7polymerase, “MT” refers to mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.) and“NT” means not tested.

TABLE 49 Combination Chemistry Protein Protein Protein Cytokines In VivoCytidine Uridine IVT IVT (Luc; (G-CSF; (G-CSF; (G-CSF; Protein analoganalog Purine Luc (G-CSF) HeLa) HeLa) PBMC) PBMC) (Luc) N4-pseudouridine A, G Pass Pass + + NT NT + acetylcytidine WT WT N4- N1- A,G Pass Pass + + NT NT + acetylcytidine methylpseu- WT WT douridine 5- 5-A, G Pass Pass + + NT NT + methylcytidine methoxyuridine WT WT 5- 5- A,G Pass Pass Not + NT NT methylcytidine bromouridine WT WT Done 5- 5- A,G Pass Pass + + NT NT + methylcytidine methyluridine WT WT 5- 50% 2- A,G Pass Pass + NT NT NT + methylcytidine thiouridine; WT WT 50% uridine5- 100% 2- A, G Pass Pass − + NT NT methylcytidine thiouridine WT WT 5-pseudouridine A, G Pass Pass + + ++ + + methylcytidine WT WT 5- N1- A, GPass Pass + + ++++ − + methylcytidine methylpseu- WT WT douridine N4-2-thiouridine A, G Not Pass Not + NT NT NT acetylcytidine Done WT DoneN4- 5- A, G Not Pass Not + NT NT NT acetylcytidine bromouridine Done WTDone N4- 2 A, G Pass Pass − + NT NT NT acetylcytidine Fluorouridinetriphosphate 5- 2 A, G Pass Pass − + NT NT NT methylcytidineFluorouridine triphosphate 2 pseudouridine A, G Pass Pass − + NT NT NTFluorocytosine triphosphate 2 N1- A, G Pass Pass − +/− NT NT NTFluorocytosine methylpseu- triphosphate douridine 2 2-thiouridine A, GPass Pass − − NT NT NT Fluorocytosine triphosphate 2 5- A, G Pass Pass −+/− NT NT NT Fluorocytosine bromouridine triphosphate 2 2 A, G Pass Pass− +/− NT NT NT Fluorocytosine Fluorouridine triphosphate triphosphate 5-uridine A, 2 Pass Pass − − NT NT NT methylcytidine Fluoro GTP 5-pseudouridine A, 2 Pass Pass − − NT NT NT methylcytidine Fluoro GTP 5-N1- A, 2 Pass Pass − +/− NT NT NT methylcytidine methylpseu- Fluorodouridine GTP 2 pseudouridine A, 2 Pass Pass − +/− NT NT NTFluorocytosine Fluoro triphosphate GTP 2 N1- A, 2 Pass Pass − − NT NT NTFluorocytosine methylpseu- Fluoro triphosphate douridine GTP

Example 90 2′Fluoro Chemistries in PBMC

The ability of G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 1;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1) to trigger innate an immune response was determined bymeasuring interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha(TNF-alpha) production. Use of in vitro PBMC cultures is an accepted wayto measure the immunostimulatory potential of oligonucleotides (Robbinset al., Oligonucleotides 2009 19:89-102) and transfection methods aredescribed herein. Shown in Table 50 are the average from 2 or 3 separatePBMC donors of the interferon-alpha (IFN-alpha) and tumor necrosisfactor alpha (TNF-alpha) production over time as measured by specificELISA. Controls of R848, P(I)P(C), LPS and Lipofectamine 2000 (L2000)were also analyzed.

With regards to innate immune recognition, while both modified mRNAchemistries largely prevented IFN-alpha and TNF-alpha productionrelative to positive controls (R848, P(I)P(C)), 2′fluoro compoundsreduce IFN-alpha and TNF-alpha production even lower than othercombinations and N4-acetylcytidine combinations raised the cytokineprofile.

TABLE 50 IFN-alpha and TNF-alpha IFN-alpha: TNF-alpha: 3 Donor 2 DonorAverage Average (pg/ml) (pg/ml) L2000 1 361 P(I)P(C) 482 544 R848 458,235 LPS 0 6,889 N4-acetylcytidine/pseudouridine 694 528N4-acetylcytidine/N1- 307 283 methylpseudouridine5-methylcytidine/5-methoxyuridine 0 411 5-methylcytidine/5-bromouridine0 270 5-methylcytidine/5-methyluridine 456 4285-methylcytidine/2-thiouridine 274 277 N4-acetylcytidine/2-thiouridine 0285 N4-acetylcytidine/5-bromouridine 44 4035-methylcytidine/pseudouridine 73 332 5-methylcytidine/N1- 31 280methylpseudouridine N4-acetylcytidine/2′fluorouridine 35 32 triphosphate5-methylcytodine/2′fluorouridine 24 0 triphosphate 2′fluorocytidinetriphosphate/N1- 0 11 methylpseudouridine 2′fluorocytidinetriphosphate/2- 0 0 thiouridine 2′fluorocytidine/triphosphate5- 12 2bromouridine 2′fluorocytidine triphosphate/ 11 0 2′fluorouridinetriphosphate 2′fluorocytidine triphosphate/5- 14 23 methylcytidine2′fluorocytidine triphosphate/5- 6 21 methylcytidine/pseudouridine2′fluorocytidine triphosphate/5- 3 15 methylcytidine/N1-methylpseudouridine 2′fluorocytidine triphosphate/ 0 4 pseudouridine2′fluorocytidine triphosphate/N1- 6 20 methylpseudouridine5-methylcytidine/pseudouridine 82 18 5-methylcytidien/N1- 35 3methylpseudouridine

Other Embodiments

It is to be understood that while the present disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the present disclosure, which is defined by the scope of the appendedclaims. Other aspects, advantages, and modifications are within thescope of the following claims.

What is claimed is:
 1. An isolated mRNA encoding a polypeptide ofinterest, said isolated mRNA comprising at least a first and a secondmodified nucleoside; wherein said first modified nucleoside isN1-methylpseudouridine and said second modified nucleoside is neitherpseudouridine or 5-methylcytosine.
 2. The isolated mRNA of claim 1,wherein a second modified nucleoside is selected from the groupconsisting of a modified purine nucleoside and a modified pyrimidinenucleoside.
 3. The isolated mRNA of claim 2, wherein the second modifiednucleoside is a modified purine nucleoside and the modified purinenucleoside is selected from the group consisting of adenosine andguanosine.
 4. The isolated mRNA of claim 3, wherein the modified purinenucleoside comprises at least one modification selected from the groupconsisting of a base modification and a sugar modification.
 5. Theisolated mRNA of claim 4, wherein the at least one modification is onthe sugar.
 6. The isolated mRNA of claim 2, wherein the modifiedpyrimidine nucleoside is selected from the group consisting of cytidineand uridine.
 7. The isolated mRNA of claim 6, wherein the modifiedpyrimidine nucleoside comprises at least one modification selected fromthe group consisting of a base modification and a sugar modification. 8.The isolated mRNA of claim 7, wherein the at least one modification ison the sugar.
 9. The isolated mRNA of claim 2 further comprising a thirdmodified nucleoside.
 10. The isolated mRNA of claim 9, wherein the thirdmodified nucleoside is selected from the group consisting of a modifiedpurine nucleoside and a modified pyrimidine nucleoside.
 11. The isolatedmRNA of claim 10, wherein the third modified nucleoside is purine andthe modified purine nucleoside comprises at least one modificationselected from the group consisting of a base modification and a sugarmodification.
 12. The isolated mRNA of claim 11, wherein the at leastone modification is on the sugar.
 13. The isolated mRNA of claim 10,wherein the third modified nucleoside is pyrimidine and the modifiedpyrimidine nucleoside comprises at least one modification selected fromthe group consisting of a base modification and a sugar modification.14. The isolated mRNA of claim 13, wherein the at least one modificationis on the sugar.